Thienopyranones as Kinase Inhibitors

ABSTRACT

The invention relates to compounds of formula I (or pharmaceutically acceptable salts thereof) as defined herein, pharmaceutical compositions thereof, and their use in manufactures and methods for modulating biological processes including inhibition of kinase activity such as PI-3 kinase.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/041,279,filed Sep. 30, 2013, which is a continuation of application Ser. No.12/735,309, filed Jul. 1, 2010, which is the National Stage ofInternational Application No. PCT/US09/31864, filed Jan. 23, 2009, whichclaims the benefit of U.S. Provisional Application No. 61/110,745, filedNov. 3, 2008 and U.S. Provisional Application No. 61/023,320, filed Jan.24, 2008, herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to new thienopyranone compounds andconjugates thereof that demonstrate activity as inhibitors of kinasesincluding PI-3 kinase, useful as anti-tumor agents in mammals.

BACKGROUND

This invention relates to thienopyranone compounds that demonstrateactivity as kinase inhibitors including PI-3 kinase. In particular, theinvention relates to new thienopyranone compounds, conjugates thereof,pharmaceutical compositions containing the thienopyranones or conjugatesthereof as active ingredients, and use of the thienopyranone compoundsas therapeutic agents including antitumor agents for the treatment ofneoplasmic disorders including cancer.

Protein kinases play an important role in regulating most cellularfunctions including proliferation, cell cycle, cell metabolism,survival/apoptosis. DNA damage repair, cell motility, and response tothe microenvironment. Not surprisingly kinases have been identified asoncogenes. For example, kinases such as c-Src, c-Abl, mitogen activatedprotein (MAP) kinase, phosphotidylinositol-3-kinase (PI3-K), AKT (alsoknown as PKB), and the epidermal growth factor (EGF) receptor arecommonly activated in cancer cells and are known to contribute totumorigenesis. Many of these mutations occur in the same signalingpathway; for example, HER-kinase family members (HER1 [EGFR], HER3, andHER4) transmit signals through MAP kinase and PI-3 kinase to promotecell proliferation.

PI-3 kinases are a large family of lipid kinases comprising roughly 16members divided into 3 classes based on sequence homology and theparticular product formed by enzyme catalysis. The class I PI-3 kinasesare composed of 2 subunits: a 110 kd catalytic subunit and an 85 kdregulatory subunit. Class 1 PI-3 kinases are involved in importantsignal transduction events downstream of cytokines, integrins, growthfactors and immunoreceptors, and control of this pathway may lead toimportant therapeutic effects. Indeed, inhibition of class I PI-3 kinaseinduces apoptosis, blocks tumor induced angiogenesis in vivo, andincreases radiosensitivity in certain tumors.

Molecular and genetic studies have demonstrated a strong correlationbetween the PI-3 kinase pathway (also known as PI3K-AKT pathway) and avariety of diseases in humans such as inflammation, autoimmuneconditions, and cancers (P. Workman et al. Nat. Biotechnol. 2006, 24,794-796). The PI-3 kinase pathway controls a number of cellularfunctions including cell growth, metabolism, differentiation, andapoptosis. Many types of cancer are thought to arise in response toabnormalities in signal transduction pathways of which the PI-3 kinasepathway is a major example. The PI-3 kinase pathway comprises a numberof enzymes including PI-3 kinase, PTEN (Phosphatase and Tensin homologdeleted on chromosome Ten), and AKT (a serine/threonine kinase) all ofwhich are involved in producing and maintaining intracellular levels ofsecond messenger molecule PtdIns(3,4,5)P3 (PIP₃). Homeostasis in thelevels of this important second messenger is maintained by theinteraction between PI-3 kinase and PTEN. When either PI-3 kinase orPTEN are mutated and/or reduced in activity PIP₃ levels are perturbedand it is believed that this perturbation may act as a trigger in thedevelopment of cancer. Indeed, both PI-3 kinase and PTEN have been foundto be mutated in multiple cancers including glioblastoma, ovarian,breast, endometrial, hepatic, melanoma, gut, lung, renal cell, thyroidand lymphoid cancer. Multiple studies have now shown that p110α, whichis a Class IA isoform of the regulatory subunit of PI-3 kinase, isfrequently over-expressed and mutated in many cancers including gliomas,colon, brain, breast, lung, prostate, gynecological and other tumortypes (Y. Samuels et al. Science 2004, 304, 554). Thus, a rationalapproach to treating cancer relates to developing drugs that act onkinases including those of the PI-3 kinase pathway.

Another putative mechanism for cancer involving kinase dependency isthrough loss of a negative regulator. Perhaps the best conceptualexample of this comes from tumors with mutations in the PTEN tumorsuppressor gene. This gene, which is mutated or deleted in a number ofdifferent cancers, encodes a lipid phosphatase that regulates signalingthrough the phosphatidylinositol 3-kinase (PI-3 kinase) pathway.Specifically. PTEN dephosphorylates PIP3, the product of PI-3 kinase(for review see L. C. Cantley et al. Proc. Natl. Acad. Sci. 1999, 96,4240-4245). As a consequence of PTEN loss and the resultant increase inPIP3 levels, signal propagation through downstream kinases such as AKTis constitutively elevated. Preclinical studies suggest that thisindirect mode of constitutive kinase activation in tumor cells, throughloss of the PTEN suppressor gene, creates a kinase dependency analogousto that seen in tumors with direct, activating mutations in the kinaseitself.

Genetic and biochemical evidence from several model systems hasestablished that constitutive levels of AKT can regulate TOR (mTOR inmammalian systems) through phosphorylation of the tuberous sclerosiscomplex (K. Inoki et al. Nat. Cell Biol. 2002, 4, 648-657). Hence,tumors with loss-of-function mutations in PTEN exhibit constitutiveactivation of AKT, as well as other downstream kinases such as mTOR.Many such tumors in murine models have been shown to be sensitive tomTOR inhibitors (M. S. Neshat et al. Proc. Natl. Acad. Sci. 2001, 98,10314-10319).

At the cytocellular level, the induction and/or progression of cancerappears to involve a sub-population of cells within a tumor known ascancer stem cells. Within a population of cancer cells there exist asmall number of cells that are capable of fully re-establishing a tumor.These cells are called cancer stem cells and are thought to beresponsible for the inability to cure cancer with current drugs. Thesecells are characterized as having enhanced drug efflux properties,lacking in cell cycle progression (quiescent), and possessing resistanceto anoikis (apoptosis upon experiencing loss of anchorage). Cancer stemcells have been described in the literature in solid tumor types forexample see the review and references incorporated therein by J. E.Visvader et al. Nat. Rev. Cancer 2008, 8, 755-768: “Cancer Stem Cells inSolid Tumors: accumulating evidence and unresolved questions”. Non-solidtumor cancer stem cells have also been reviewed recently, for example,see the review and references incorporated therein by J. E. Dick et al.Blood 2008, 4793-4807: “Stem cell concepts renew cancer research”. Todate the only documented clinical example of an approved cancertherapeutic drug decreasing cancer stem cells has been the use ofLapatinib shown to decrease the number of breast cancer stem cells inbiopsies of women with breast tumors possessing high levels of HER2protein (decreased from 11% down to 5% of cells) [C. Schmidt et al. J.Natl. Cancer I. 2008, 100, 694-695: “Lapatinib Study Supports CancerStem Cell Hypothesis, Encourages Industry Research”].

While therapeutic agents that act as modulators of signaling pathwaysare of clear therapeutic interest as agonists or antagonist ofparticular enzymes within a signaling pathway, e.g. inhibitors of PI-3kinase, recent evidence indicates that independent mechanisms exist forproviding therapeutic efficacy including, for example, oxidative stress.The generation of oxidative stress in cancer cells is a recent but welldescribed cancer treatment approach. Examples of agents that induce suchstress include clinically evaluated compounds such as buthioninesulfoximine/melphalan, imexon, arsenic trioxide, and motexafingadolinium and the like [see for example the review and referencesincorporated therein by R. H. Engel et al. Front. Biosci 2006, 11,300-312: “Oxidative Stress and Apoptosis: a new treatment paradigm incancer”]. Cromenones such as LY294002 and the related analog LY3035111have been reported to induce apoptosis in tumor cells due tointracellular hydrogen peroxide production independent of their PI3kinase inhibition activity [T. W. Poh et al. Cancer Res. 2005,6264-6274. “LY294002 and LY303511 Sensitize Tumor Cells to Drug-InducedApoptosis via Intracellular Hydrogen Peroxide Production Independent ofthe Phosphoinositide 3-Kinase-Akt Pathway”]. This ability to induceoxidative stress in cancer cells is a positive attribute for ananticancer agent. Oxidative stress induction has also been demonstratedto enhance sensitivity of prostate cancer cells to nonapopototicconcentrations of the chemotherapeutic agent vincristine.

LY294002 (2-(4-morpholinyl)-K-phenyl-4H-1-benzopyran-4-one) is a potent,non-selective inhibitor of PI-3 kinases with an IC50 of 1.4 μM (C. J.Vlahos et al. J. Biol. Chem. 1994, 269, 5241-5248). While LY294002 is aneffective inhibitor of PI-3 kinase it has several undesirable attributesfor clinical use including lack of aqueous solubility, poorpharmacokinetics, unacceptable toxicity, no tissue specificity, rapidmetabolism in animals, and a synthetic route that involves the use ofcarbon disulfide, a highly toxic compound. As such, LY294002 has neverbeen de-eloped for clinical use.

Thus, there continues to be a need for safe and effective kinaseinhibitors, including Pt-3 kinase inhibitors, that are suitable forclinical use as tumor growth inhibitors and cancer stem cell inhibitors.The present invention relates to thienopyranone compounds thatsurprisingly provide significantly improved properties and advantagesover LY294002 including unexpectedly superior potency as PI-3 kinase,cancer cell and cancer stem cell inhibitors, oxidative stress inducers,and synthetic procedures that avoid the need to use highly toxiccompounds such as carbon disulfide.

SUMMARY OF THE INVENTION

The present invention relates in one aspect to thienopyranone(7H-thieno[3,2-b]pyran-7-ones) compounds of the Formula I or apharmaceutically acceptable salt thereof:

wherein M is oxygen (O) or sulfur (S);

R1 is selected from H, halogen, alkyl, alkenyl, alkynyl, carbocycle,aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino, carboxylicacid, carboxylic ester, carboxyl amide, reverse carboxyamide,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedcarbocycle, substituted aryl, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate:R2 is selected from R1 or

where X is C, N, P, P(O), SiR^(b);n is 0, 1, or 2;Y is C—R1, O, S, NR^(a), —C(O)(NH₂), —P(Z)_(m)R³, SiR^(a)R^(b), BR^(b);

Z is O or S;

m=0 or 1;R^(a) is hydrogen (H) or independently at each instance any groupdefined in R1;R^(b) is hydrogen (H) or independently at each instance any groupdefined in R1;R3 is selected from R1:R4 is selected from R1; andCyc is an aryl, substituted aryl, heterocycle, substituted heterocycle,carbocycle, and substituted carbocycle.

As used herein, the expression “a compound of Formula X” (e.g. FormulaI, II, III, etc.), or the expression “a compound of the invention”includes the compound, conjugates thereof, and any conventional prodrugthereof, as well as a pharmaceutically acceptable salt of said compound,conjugate, or prodrug. The compounds of the present invention alsoencompass polymorphic forms, solvates, hydrates, salts and complexesthereof.

Compounds of Formula I are useful as inhibitors of kinases including,for example and not limited to, mTOR kinase, PIM-1 kinase, PLK-1 kinase,DNA-PK kinase, and Class IA and IB PI-3 kinases. In addition, variouscompounds of Formula I are useful inhibitors of tumor growth and for thetreatment of cancer.

Accordingly, it is an object of the present invention to providecompounds, compositions, and methods for inhibiting kinases, for examplePI-3 kinases, and for inhibiting cancerous tumor growth and causingtumor reduction.

These and other objects of the invention are evidenced by the summary ofthe invention, the description of the preferred embodiments and theclaims.

DETAILED DESCRIPTION A. Definitions

“Cancer” refers to cellular-proliferative disease states, including butnot limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hanlartoma, mesothelioma: Gastrointestinal: esophagus(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors.Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma): Genitourinary tract: kidney (adenocarcinoma,Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma,embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors,lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma: Bone:osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewings sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors, Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependyrnoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schiwannoma, retinoblastoma congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma): Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors. Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma[embryonal rhabdomyosarcoma], fallopian tubes (carcinoma): Hematologic:blood (myeloid leukemia [acute and chronic], acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, myelodysplastic syndrome), Hodgkin's disease,non-Hodgkin's lymphoma [malignant lymphoma]: Skin: malignant melanoma,basal cell carcinoma, squamous cell carcinoma. Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis;Adrenal Glands: neuroblastoma; and breast cancer.

The term “cancerous cell” as provided herein, includes a cell affectedby any one of the above-identified cancers. The term “cancer stem cell”refers to a subpopulation of cells in a solid or non-solid tumor thatdemonstrate enhanced drug efflux properties, are lacking in cell cycleprogression, and are resistant to anoikis.

As used herein, the term “branched” refers to a group containing from 1to 24 backbone atoms wherein the backbone chain of the group containsone or more subordinate branches from the main chain. Preferred branchedgroups herein contain from 1 to 12 backbone atoms. Examples of branchedgroups include, but are not limited to, isobutyl, t-butyl, isopropyl.—CH₂CH₂CH(CH₃)CH₂CH₃, —CH₂CH(CH₂CH₃)CH₂CH₃, —CH₂CH₂C(CH₃)₂CH₃,—CH₂CH₂C(CH₃)₃, and the like.

The term “unbranched” as used herein refers to a group containing from 1to 24 backbone atoms wherein the backbone chain of the group extends ina direct line. Preferred unbranched groups herein contain from 1 to 12backbone atoms.

The term “cyclic” or “cyclo” as used herein alone or in combinationrefers to a group having one or more closed rings, whether unsaturatedor saturated, possessing rings of from 3 to 12 backbone atoms,preferably 3 to 7 backbone atoms.

The term “lower” as used herein refers to a group with 1 to 6 backboneatoms.

The term “saturated” as used herein refers to a group where allavailable valence bonds of the backbone atoms are attached to otheratoms. Representative examples of saturated groups include, but are notlimited to, butyl cyclohexyl, piperidine and the like.

The term “unsaturated” as used herein refers to a group where at leastone available valence bond of two adjacent backbone atoms is notattached to other atoms. Representative examples of unsaturated groupsinclude, but are not limited to, —CH₂CH₂CH═CH₂, phenyl, pyrrole and thelike.

The term “aliphatic” as used herein refers to an unbranched, branched orcyclic hydrocarbon group, which may be substituted or unsubstituted, andwhich may be saturated or unsaturated, but which is not aromatic. Theterm aliphatic further includes aliphatic groups, which comprise oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone.

The term “aromatic” as used herein refers to an unsaturated cyclichydrocarbon group which may be substituted or unsubstituted having 4n+2delocalized π(pi) electrons. The term aromatic further includes aromaticgroups, which comprise a nitrogen atom replacing one or more carbons ofthe hydrocarbon backbone. Examples of aromatic groups include, but arenot limited to, phenyl, naphthyl, thienyl, furanyl, pyridinyl,(is)oxazoyl and the like.

The term “substituted” as used herein refers to a group having one ormore hydrogens or other atoms removed from a carbon or suitableheteroatom and replaced with a further group. Preferred substitutedgroups herein are substituted with one to five, most preferably one tothree substituents. An atom with two substituents is denoted with “di,”whereas an atom with more than two substituents is denoted by “poly.”Representative examples of such substituents include, but are notlimited to aliphatic groups, aromatic groups, alkyl, alkenyl, alkynyl,aryl, alkoxy, halo, aryloxy, carbonyl, acryl, cyano, amino, nitro,phosphate-containing groups, sulfur-containing groups, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, alkylcarbon, arylcarbonyl, acarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, acylamino, amidino, imino, alkylthio, arylthio,thiocarboxylate, alkylsulfinyl, trifluoromethyl, azido, heterocyclyl,alkylaryl, heteroaryl, semicarbazido, thiosemicarbazido, maleimido,oximino, imidate, cycloalkyl, cycloalkylcarbonyl, dialkylamino,arylcycloalkyl, arylcarbonyl, arylalkylcarbonyl, arylcycloalkylcarbonyl,arylphosphinyl, arylalkylphosphinyl, arylcycloalkylphosphinyl,arylphosphonyl, arylalkylphosphonyl, arylcycloalkylphosphonyl,arylsulfonyl, arylalkylsulfonyl, arylcycloalkylsulfonyl, combinationsthereof, and substitutions thereto.

The term “unsubstituted” as used herein refers to a group that does nothave any further groups attached thereto or substituted therefore.

The term “alkyl” as used herein, alone or in combination, refers to abranched or unbranched, saturated aliphatic group. The alkyl radical maybe optionally substituted independently with one or more substituentsdescribed herein. Lower alkyl refers to alkyl groups of from one to sixcarbon atoms. Examples of lower alkyl groups include methyl, ethyl,propyl, isopropyl, butyl, s-butyl, t-butyl, isobutyl, pentyl, and thelike. Higher alkyl refers to alkyl groups containing more than sevencarbon atoms. A “Co” alkyl (as in “Co—C₆-alkyl”) is a covalent bond.Exemplary alkyl groups are those of C₂₀ or below. In this application,alkyl refers to alkanyl, alkenyl, and alkynyl residues (and combinationsthereof); it is intended to include vinyl, allyl, isoprenyl, and thelike. Thus when an alkyl residue having a specific number of carbons isnamed, all geometric isomers having that number of carbons are intendedto be encompassed: thus, for example, either “butyl” or “C₄ alkyl” ismeant to include n-butyl, sec-butyl, isobutyl, t-butyl, isobutenyl andbut-2-ynyl groups; and for example, “propyl” or “C₃ alkyl” each includen-propyl, propenyl, and isopropyl. Representative examples of alkylgroups include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. The terms“alkyl” or “alk” as used herein refer to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C.sub.1-C.sub.12), wherein the alkyl radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkyl radical is one to eight carbon atoms(C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples of alkyl groupsinclude, but are not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃),1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl,—CH(CH₃)₂), 1-butyl (n-Bu, n-butyl. —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl(i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)₂),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The terms “carbocycle”. “carbocyclyl”. “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C.sub.3-C.sub.12) as amonocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Thecycloalkyl radical may be optionally substituted independently with oneor more substituents described herein. Bicyclic carbocycles having 7 to12 atoms can be arranged, for example, as a bicyclo[4,5], [5,5], [5,6]or [6,6] system, and bicyclic carbocycles having 9 or 10 ring atoms canbe arranged as a bicyclo[5,6] or [6,6] system, or as bridged systemssuch as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

The term “alkenyl” as used herein alone or in combination refers to abranched or unbranched, unsaturated aliphatic group containing at leastone carbon-carbon double bond which may occur at any stable point alongthe chain. The alkenyl radical may be optionally substitutedindependently with one or more substituents described herein, andincludes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Representative examples ofalkenyl groups include, but are not limited to, ethenyl, E- andZ-pentenyl, decenyl and the like.

The term “alkynyl” as used herein alone or in combination refers to abranched or unbranched, unsaturated aliphatic group containing at leastone carbon-carbon triple bond which may occur at any stable point alongthe chain. The alkynyl radical may be optionally substitutedindependently with one or more substituents described herein.Representative examples of alkynyl groups include, but are not limitedto, ethynyl, propynyl, propargyl, butynyl, hexynyl, decynyl and thelike.

The term “aryl” as used herein alone or in combination refers to asubstituted or unsubstituted aromatic group, which may be optionallyfused to other aromatic or non-aromatic cyclic groups. Aryl includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typical arylgroups include, but are not limited to, radicals derived from benzene(phenyl), substituted benzenes, naphthalene, anthracene, biphenyl,indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl,and the like. Aryl groups are optionally substituted independently withone or more substituents described herein.

The term “alkoxy” as used herein alone or in combination refers to analkyl, alkenyl or alkynyl group bound through a single terminal etherlinkage. Examples of alkoxy groups include, but are not limited to,methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, 2-butoxy,tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy,n-hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, fluoromethoxy,difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, andtrichloromethoxy.

The term “aryloxy” as used herein alone or in combination refers to anaryl group bound through a single terminal ether linkage.

The terms “halogen”, “halo” and “hal” as used herein refer to monovalentatoms of fluorine, chlorine, bromine, iodine and astatine.

The term “hetero” or “heteroatom” as used herein combination refers to agroup that includes one or more atoms of any element other than carbonor hydrogen. Representative examples of hetero groups include, but arenot limited to, those groups that contain heteroatoms including, but notlimited to, nitrogen, oxygen, sulfur and phosphorus.

The term “heterocycle” or “heterocyclyl” or “heterocyclic ring” or“heterocyclic” as used herein refers to a cyclic group containing one ormore heteroatoms. The heterocyclic radical may be optionally substitutedindependently with one or more substituents described herein.

Representative examples of heterocycles include, but are not limited to,pyridine, piperadine, pyrimidine, pyridazine, piperazine, pyrrole,pyrrolidinone, pyrrolidine, morpholine, thiomorpholine, indole,isoindole, imidazole, triazole, tetrazole, furan, benzofuran,dibenzofuran, thiophene, thiazole, benzothiazole, benzoxazole,benzothiophene, quinoline, isoquinoline, azapine, naphthopyran,furanobenzopyranone and the like.

The term “substituent” means any group selected from H. F, Cl, Br, I,alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocycle, heteroaryl,formyl, nitro, cyano, amino, carboxylic acid, carboxylic ester, carboxylamide, reverse caboxyamide, halo, haloalkyl, haloalkoxy, hydroxy, oxo(valency rules permitting), lower alkanyl, lower alkenyl, lower alkynyl,alkoxy, optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, alkylaminoalkyl, dialkylaminoalkyl, carboxy, carboxy ester.—C(O)NR⁵R″ (where R⁵ is hydrogen or alkyl and R″ is hydrogen, alkyl,aryl, or heterocyclyl), —NR⁵C(O)R″ (where R³ is hydrogen or alkyl and R″is alkyl, aryl, or heterocyclyl), amino, alkylamino, dialkylamino, and—NHS(O)₂R′ (where R′ is alkyl, aryl, or heteroaryl).

The term “carbonyl” or “carboxy” as used herein alone or in combinationrefers to a group that contains a carbon-oxygen double bond.Representative examples of groups which contain a carbonyl include, butare not limited to, aldehydes (i.e., formyls), ketones (i.e., acyls),carboxylic acids (i.e., carboxyls), amides (i.e., amidos), imides (i.e.,imidos), esters, anhydrides and the like.

The term “carbamate” as used herein alone or in combination refers to anester group represented by the general structure —NH(CO)O—. Carbamateesters may have alkyl or aryl groups substituted on the nitrogen, or theamide function.

term “cyanate” “isocyanate”. “thiocyanate”, or “isothiocyanate” as usedherein alone or in combination refers to an oxygen- or sulfur-carbondouble bond carbon-nitrogen double bond. Representative examples ofcyano groups include, but are not limited to, isocyanate, isothiocyanateand the like.

The term “cyano”, “cyanide”, “isocyanide”, “nitrile”, or “isonitrile” asused herein alone or in combination refers to a carbon-nitrogen triplebond.

The term “amino” as used herein alone or in combination refers to agroup containing a backbone nitrogen atom. Representative examples ofamino groups include, but are not limited to, alkylamino, dialkylamino,arylamino, diarylamino, alkylarylamino, alkylcarbonylamino,arylcarbonylamino, carbamoyl, ureido and the like.

The term “phosphate-containing group” as used herein refers to a groupcontaining at least one phosphorous atom in an oxidized state.Representative examples include, but are not limited to, phosphonicacids, phosphinic acids, phosphate esters, phosphinidenes, phosphinos,phosphinyls, phosphinylidenes, phosphos, phosphonos, phosphoranyls,phosphoranylidenes, phosphorosos and the like.

The term “sulfur-containing group” as used herein refers to a groupcontaining a sulfur atom. Representative examples include, but are notlimited to, sulfhydryls, sulfenos, sulfinos, sulfinyls, sulfos,sulfonyls, thios, thioxos and the like.

The term “optional” or “optionally” as used herein means that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not. For example, the phrase“optionally substituted alkyl” means that the alkyl group may or may notbe substituted and that the description includes both unsubstitutedalkyl and substituted alkyl.

The term “targeting agent” as used herein means any moiety whoseattachment to a compound of the invention allows the increase inconcentration of the compound at a site of treatment, for example, atumor site. Exemplary targeting agents include but are not limited tocarbohydrates, peptides, vitamins, and antibodies.

The term “effective amount” or “effective concentration” when used inreference to a compound, product, or composition as provided herein,means a sufficient amount of the compound, product or composition toprovide the desired pharmaceutical or therapeutic result. The exactamount required will vary depending on the particular compound, productor composition used, its mode of administration and the like. Thus, itis not always possible to specify an exact “effective amount.” However,an appropriate effective amount may be determined by one of ordinaryskill in the art informed by the instant disclosure using only routineexperimentation.

The term “hydrolyzable” as used herein refers to whether the group iscapable of or prone to hydrolysis (i.e., splitting of the molecule orgroup into two or more new molecules or groups).

The term “pharmaceutically acceptable salt” of a compound of the instantinvention (e.g. Formula I) is one which is the acid addition salt of abasic compound of the invention with an inorganic or organic acid whichaffords a physiologically acceptable anion or which is the salt formedby an acidic compound of the invention with a base which affords aphysiologically acceptable cation.

The term “prodrug” or “procompound” as used in this application refersto a precursor or derivative form of a compound of the invention thatmay be less cytotoxic to cells compared to the parent compound or drugand is capable of being enzymatically or hydrolytically activated orconverted into the more active parent form. See, e.g., Wilman, “Prodrugsin Cancer Chemotherapy” Biochemical Society Transactions, 14, pp.375-382, 615th Meeting Belfast (1986) and Stella et al., “Prodrugs: AChemical Approach to Targeted Drug Delivery,” Directed Drug Delivery,Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugsof this invention include, but are not limited to, phosphate-containingprodrugs, thiophosphate-containing prodrugs, sulfate-containingprodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, beta-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs, optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, compounds of the invention and chemotherapeutic agents suchas described above.

The term “conjugate” as used herein refers to a compound that has beenformed by the joining of two or more compounds via either a covalent ornon-covalent bond.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate” ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, trifluoroacetic acid, maleic acid, succinicacid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalicacid, glycolic acid, salicylic acid, a pyranosidyl acid, such asglucuronic acid or galacturonic acid, an alpha hydroxy acid, such ascitric acid or tartaric acid, an amino acid, such as aspartic acid orglutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid,a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid,or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperadine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.The term “hydrate” refers to the complex where the solvent molecule iswater.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and9-fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a “hydroxy-protectinggroup” refers to a substituent of a hydroxy group that blocks orprotects the hydroxy functionality. Suitable protecting groups includeacetyl and silyl. A “carboxy-protecting group” refers to a substituentof the carboxy group that blocks or protects the carboxy functionality.Common carboxy-protecting groups include phenylsulfonylethyl,cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl,2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl,2-(diphenylphosphino)-ethyl, nitroethyl and the like. For a generaldescription of protecting groups and their use, see T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,1991.

The terms “compound of this invention.” and “compounds of the presentinvention” include compounds of Formulas I-IX and stereoisomers,geometric isomers, tautomers, solvates, metabolites, andpharmaceutically acceptable salts, prodrugs, and conjugates thereof.

B. Compounds

The present invention pros ides in part compounds of the Formula I:

wherein M is O or S:R1 is selected from H, F, Cl, Br, I, alkyl, alkenyl, alkynyl,carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino,carboxylic acid, carboxylic ester, carboxyl amide, reverse caboxyamide,substituted alkyl, substituted alkenyl, substituted alkynyl substitutedcarbocycle, substituted aryl, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid. O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide. N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate:R2 is selected from R1 or

Where X is C, N, P, P(O), SiR^(b);

n is 0, 1, or 2;Y is C—R1, O, S, NR^(a), —C(O)(NH₂), —P(Z)_(m)R^(a), SiR^(a)R^(b),BR^(b);

Z is O or S;

m=0 or 1;R^(a) is hydrogen (H) or independently at each instance any groupdefined in R1;R^(b) is hydrogen (H) or independently at each instance any groupdefined in R1;R3 is selected from R1:R4 is selected from R1; andCyc is an aryl, substituted aryl, heterocycle, substituted heterocycle,carbocycle, and substituted carbocycle.

A particular compound of Formula I is one wherein a substitutent of R1comprises a bone directing group such as, for example, amino phosphonicacid, bisphsphonate, or the like. The present invention also providescompounds of Formulas II-IV:

wherein M is O or S;R1 is selected from H, F, Cl, Br, I, alkyl, alkenyl, alkynyl,carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino,carboxylic acid, carboxylic ester, carboxyl amide, reverse carboxyamide,substituted alkyl, substituted alkenyl), substituted alkynyl,substituted carbocycle, substituted aryl, substituted heterocycle,substituted heteroaryl, phosphonic acid, phosphinic acid,phosphoramidate, phosphonic ester, phosphinic ester, ketone, substitutedketone, hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate.R3 is independently, at each instance, R1, andR4 is independently, at each instance, R1.

A particular compound of Formulas II-IV is one wherein a substitutent ofR1 comprises a bone directing group such as, for example, aminophosphonic acid, bisphsphonate, or the like.

The present invention also provides compounds of Formulas V-VII:

wherein R3 is selected from H, F, Cl, Br, I, alkyl, alkenyl, alkynyl,carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino,carboxylic acid, carboxylic ester, carboxyl amide, reverse caboxyamide,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedcarbocycle, substituted aryl, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate. N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide. N-substituted sulfonamide. N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate.

A particular compound of Formulas V-VII is one wherein a substitutent ofR3 comprises a bone directing group such as, for example, aminophosphonic acid, bisphsphonate, or the like. (See exemplary compounds inTable 8).

C. Conjugates

The present invention also provides conjugates of Formula I. In oneembodiment conjugates are formed by alkylating a compound of Formula Iwith a linker group (L), the linker group optionally being substitutedwith a targeting agent (T). Methods for producing conjugates for thisaspect of the invention include alkylation procedures disclosed in U.S.Pat. No. 6,949,537 and U.S. Pat. No. 7,396,828 the entire contents ofwhich is herein incorporated by reference. In one embodiment of thisaspect of the invention a compound of Formula I is reacted with ahalomethyl ester compound of Formula Q:

wherein Hal is a halogen: R5 is CH₂, CH(CH₃), CH(Ph), C(CH₃)COOH), orCH(CH(CH₃)₂); Z1 and Z2 are independently S or O;R6 is hydrogen, optionally substituted aliphatic, optionally substitutedaryl, alkoxy, carboxy, amino, heterocycle, aryloxy, and optionallysubstituted therewith a targeting agent (T) to form R6-T.

Targeting Agent.

In another embodiment, conjugates of the present invention are thosecompounds wherein, R₆ further comprises one or more targeting agents (T)covalently attached thereto. Targeting agents allow the conjugates ofthe present invention to be delivered selectively to specific types ofcells, tissues, organs or extracellular structures such as receptors. Insome applications it may be desirable to limit the location of a drug orprodrug to the area of treatment or at least prevent it from reachingtissues where it can cause undesirable side effects, and to ensure thatat any particular time effective, but not excessive, amounts of the drugare used. The use of targeting agents may allo the conjugates of thepresent invention to be concentrated at the site of treatment. Oncedelivered to a site of treatment, the linker may be enzymaticallycleaved or hydrolyzed to yield a compound of formula I. Moreover, theuse of a targeting agent may limit the dosage required to achieve aneffective concentration of a drug at the site of treatment. The use oftargeting agents may also reduce the frequency of dosages required.

Suitable targeting agents are preferentially attached to compounds ofthe present invention via a covalent bond which may be formed by methodsincluding, but not limited to, a nucleophilic or electrophilic group ofthe targeting agent that is covalently reacted with an electrophilic ornucleophilic group (respectively) on the linker. In one embodiment,suitable targeting agents are those disclosed in U.S. Pat. No.6,949,537, the entire contents of which is herein incorporated byreference.

In one embodiment of the present invention, conjugates of the presentinvention are those compounds wherein. R6-T is selected from the groupconsisting of the following:

Targeting agents which may be reacted with the conjugates of the presentinvention include, but are not limited to, carbohydrates, vitamins,peptides, proteins, nucleosides, nucleotides, nucleic acids, liposomes,lipids, bone-seeking agents and cartilage-seeking agents. The targetingagent may also be a molecule which is bound by a receptor in a desiredtissue and optionally transported into a cell by a receptor-mediatedprocess. Representative examples of such targeting agents include, butare not limited to, diazepines that bind to peripheral benzodiazepinereceptors (PBRs) present in glial cells in the brain. Representativeexamples of such diazepines are discussed in G. Trapani et al.Bioconjugate Chem. 2003, 14, 830-839 entitled “Peripheral BenzodiazepineReceptor Ligand-Melphalan Conjugates for Potential Selective DrugDelivery to Brain Tumors,” the contents of which are incorporated byreference.

Representative vitamins that may be used as targeting agents include,but are not limited to, folate, vitamin B.sub.12 or vitamin C. The term“folate” encompasses folic acid derivatives with capacity to bind withfolate-receptors. Representative examples of folates that may be used astargeting agents include, but are not limited to, folic acid, folinicacid, pteropolyglutamic acid, and folate receptor-binding pteridinessuch as tetrahydropterins, dihydrofolates, tetrahydrofolates and theirdeaza and dideaza analogs. Other suitable folates are folate analogsincluding, but not limited to, aminopterin, amethopterin (methotrexate),N.sub.10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as1-deazamethopterin or 3-deaxamethopterin, and3′5′-dichloro4-amino-4-deoxy-N.sub.10-methylpteroyl-glutamic acid(dichloromethotrexate). Methods of conjugating molecules to folates thatare suitable for covalent attachment to compounds of the presentinvention are disclosed in U.S. Pat. Nos. 6,576,239, 5,820,847,5,688,488, 5,108,921, 5,635,382, and 5,416,016 the contents of which areincorporated herein by reference. Methods of conjugating molecules tovitamin C that are suitable for covalent attachment of compounds of thepresent invention are disclosed in S. Manfrdini et al. J. Med. Chem.2002, 45, 559-562, the contents of which are incorporated herein byreference.

Representative peptides and peptidomimetics that may be used astargeting agents include, but are not limited to, an RGD-containingpeptide selected from the group consisting of RGDs, c(RGDfK),vitronectin, fibronectin, somatostatin-receptor agonists andsomatostatin-receptor antagonists. Molecules that bind to the avb3integrin receptor and act as antagonists may be used as targeting agentsare described in U.S. Pat. Nos. 6,552,079, 6,426,353B, WO 2002/40505A2,and U.S. Patent Publications 2002/0055499, 2002/0061885, 2002/0065291,2002/0072500, U.S. 2002/0072518; W. Arap et al. Science 1998, 279,377-380: R. J. Kok et al Bioconjugate Chem. 2002, 13, 128-135: D. A.Sipkins et al. Nat. Med. 1998, 4, 623-626; P. M. Winter et al. CancerRes. 2003, 63, 5838-5843; and J. D. Hood et al. Science 2002, 296,2404-2407, the contents of which are incorporated herein by reference.Representative proteins that may be used as targeting agents include,but are not limited to, antibodies or fragments thereof, such as atumor-specific monoclonal antibody or fragment thereof. Representativebone-seeking agents that may be used as targeting agents include, butare not limited to, phosphonate, phosphonic acid, aminomethylphosphonicacid, phosphate, polyphosphate, and hydroxyapatite-binding polypeptides.Other peptides include chlorotoxin (U.S. Pat. No. 6,429,187B1) andtissue factor (G. M. Lanza et al. Circulation 2002, 106, 2842-2847).

Other suitable targeting agents include antibodies. The antibodies maybe of classes IgG, IgM, IgA, IgD or IgE, or fragments or derivatisesthereof, including Fab, F(ab′).sub.2, Fd, and single chain antibodies,diabodies, bispecific antibodies, bifunctional antibodies andderivatives thereof. The antibody may be a monoclonal antibody,polyclonal antibody, affinity purified antibody, or mixtures thereofwhich exhibits sufficient binding specificity to a desired epitope or asequence derived therefrom. The antibodies may also be a chimericantibody. The antibodies may be directed against a variety of antigenicdeterminants including those associated with tumors, histocompatibilityand other cell surface antigens, bacteria, fungi, viruses, enzymes,toxins, drugs and other biologically active molecules. Antigensassociated with tumors for which antibodies may be specifically reactiveinclude, but are not limited to, such antigens as are and include, butare not limited to, carcinoembryonic antigen (CEA), mucins such asTAG-72, human milk fat globule antigens, prostate serum antigent (PSA),prostate specific membrane antigen (PSMA), PS (phosphatidyl serine), andreceptors including, but not limited to, the IL-2, EGF. VEGF andtransferrin receptors. Other representative antigens associated withtumors include, but are not limited to, those tumor associated antigensdescribed in J. R. Zalcberg et al. J. Clin. Oncology 1985, 3, 876-882,WO 01/68709A1, and U.S. Patent Publication US2004/0009122A1, thecontents of which are incorporated herein by reference.

Other suitable targeting agents include glucose, galactose, mannose,mannose 6-phosphate, hormones (e.g., insulin, growth hormone, and thelike), growth factors or cytokines (e.g., TGF.beta., EGF, insulin-likegrowth factor, and the like), YEE(GalNAcAH).sub.3 or derivatives,cobalamin, .alpha.-2 macroglobulins, asialoglycoprotein, albumin,texaphyrin, metallotexaphyrin, antibodies, antibody fragments (e g.Fab), single-chain antibody variable region (scFv), transferrin, anyvitamin and any coenzyme.

The targeting agent may also be an agent that delivers a compound of theinvention to bones. Bone targeting agents include, but are not limitedto, bisphosphonates, EDTMP DOTMP, and ABEDTMP, which are disclosed inU.S. Pat. Nos. 4,937,333, 4,882,142, 5,064,633 and WO-94/00143, thecontents of which are incorporated herein by reference. DOTMP and EDTMPmay be attached to the linker moiety by any suitable coupling methodincluding, but not limited to, coupling chemistry where the R group canhave an appropriate electrophilic or nucleophilic group that reacts withthe nucleophilic or electrophilic (respectively) group of the linkermoiety. Further details of the coupling chemistry are provided inTetrahedron 1999, 55, 12997-13010, the contents of which areincorporated by reference. Further details of bone-targeted prodrugs andcoupling chemistry are provided in Proc. SPIE-Int. Soc. Opt. Eng. 1999,3600 (Biomedical Imagn. Reporters Dyes & Instrumental, 99-106. U.S. Pat.No. 5,177,054; J. Med. Chem. 1994, 37, 498-511; Tetrahedron Lett. 1989,30, 7141-7144; T. J. Houghton et al. J. Med Chem. 2008, 51, 6955-6969;and U.S. Pat. No. 5,955,453, the contents of which are incorporated byreference.

The targeting agent may be used to deliver a conjugate of the invention(or salt thereof) to bones as a slow release reservoir site for thecompounds of the present invention. The targeting agent may be a boneseeking (osteotropic) moiety attached to the compounds of the presentinvention via an acid cleavable linker. Examples of an acid cleavablelinker include, but are not limited to, an ortho acid-amide linkage.Under acidic conditions the protein-ACL-3 amide linkage is readilycleaved freeing the native amino group of the amide functionality asdescribed in WO-94/00143 the contents of which are incorporated byreference. During osteoclastic bone resorption, which involves an acidicmediated mechanism, the attachment tethering the prodrug to bone may becleaved releasing the compounds of the present invention. Methods andparticular bone-targeting agents are disclosed in U.S. Pat. No.6,949,537, the entire contents of which are herein incorporated byreference.

The targeting agent may also comprise an RGD peptide moiety. Asdiscussed in F. Curnis et al. Cancer Res. 2004, 64, 565-571. RGDmoieties target RGD fusion proteins to vasculature by interacting withcell adhesion receptors, including aνβ₃ integrin.

Conjugate compounds according to this aspect of the invention aredepicted by Formula VIII or Formula IX wherein a hydrolyzable linker Rcis in either of two positions (as shown).

wherein R1, R3, and R4 independently represent H, F, Cl, Br, I, alkyl,alkenyl, alkynyl, carbocycle, aryl, heterocycle, heteroaryl, formyl,nitro, cyano, amino, carboxylic acid, carboxylic ester, carboxyl amide,reverse carboxyamide, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted carbocycle, substituted aryl,substituted heterocycle, substituted heteroaryl, phosphonic acid,phosphinic acid, phosphoramidate, phosphonic ester, phosphinic ester,ketone, substituted ketone, hydroxamic acid, N-substituted hydroxamicacid, O-substituted hydroxamate. N- and O-substituted hydroxamate,sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfonicacid, sulfonic ester, sulfonamide, N-substituted sulfonamide,N,N-disubstituted sulfonamide, boronic acid, boronic ester, azo,substituted azo, azido, nitroso, imino, substituted imino, oxime,substituted oxime, alkoxy, substituted alkoxy, aryloxy, substitutedaryloxy, thioether, substituted thioether, carbamate, substitutedcarbamate; Rc comprises a hydrolyzable linker group (L) which isoptionally substituted with a targeting agent (T).

Exemplary (but not limiting) compounds of Formula I from whichconjugates according to this aspect of the invention can be madefollowing the procedures disclosed herein are those compounds depictedin Table 8. In one embodiment, a targeted conjugate of Formula I is onein which Rc has the structure:

A pharmaceutically acceptable salt of a compound of the instantinvention is one which is the acid addition salt of a basic compound offormula I with an inorganic or organic acid which affords aphysiologically acceptable anion or which is the salt formed by anacidic compound of formula I with a base which affords a physiologicallyacceptable cation and provides a particular aspect of the invention.Examples of such acids and bases are provided hereinbelow.

As an additional aspect of the invention there is provided apharmaceutical formulation comprising in association with apharmaceutically acceptable carrier, diluent or excipient, a compound offormula I, VIII, and IX (or a pharmaceutically acceptable salt thereof)as provided in any of the descriptions herein.

In addition, compounds (or salts thereof) of the present invention areuseful as an active ingredient in the manufacture of a medicament foruse in inhibiting kinase activity e.g. PI-3 kinase activity.

The present invention also provides a method of inhibiting kinaseactivity in a mammal comprising administering to a mammal in need oftreatment, a kinase inhibiting dose of a compound of formula I orconjugate or prodrug thereof having any of the definitions herein.

The present invention further provides a method of inhibiting PT-3kinase comprising administering to a mammal in need of treatment, a PI-3kinase-inhibiting dose of a compound of formula I or conjugate orprodrug thereof having any of the definitions herein.

Further, the present invention provides a method of inhibiting tumorgrowth comprising administering to a mammal in need of treatment, aneffective dose of a compound of formula I, or conjugate or prodrugthereof having any of the definitions herein.

Also, there is provided a compound of formula I (or conjugate, prodrug,or salt thereof) having any of the definitions herein for use as ananticancer agent.

In addition, there is provided use of a compound of formula I having anyof the definitions herein for the manufacture of a medicament, includinga medicament for treatment of cancer.

As an additional feature of the invention there is provided apharmaceutical formulation comprising in association with apharmaceutically acceptable carrier, diluent or excipient, a conjugateof a compound of formula I (or of a pharmaceutically acceptable saltthereof) as provided in any of the descriptions herein.

The present invention also includes isotopically-labeled compounds, andpharmaceutically acceptable salts thereof, which are identical to thoserecited in Formulas I through IX, but replace one or more atoms by acorresponding isotope. Examples of isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorous, fluorine and chlorine. Compounds of thepresent disclosure, conjugates thereof, and pharmaceutically acceptablesalts of said compounds or of said conjugates which contain theaforementioned isotopes and/or other isotopes of other atoms are withinthe scope of this disclosure. Certain isotopically-labeled compounds ofthe present disclosure, for example those into which radioactiveisotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/orsubstrate tissue distribution assays for example when imaging tumors.Fluorine-18 (¹⁸F) is particularly preferred for ease of preparation anddetectability. Isotopically labeled compounds of the invention cangenerally be prepared by carrying out the procedures disclosed in theSchemes and/or in the Examples and Preparations below, by substituting areadily available isotopically labeled reagent for a non-isotopicallylabeled reagent.

It will be appreciated that certain compounds of formula I (or salts,procompounds, or conjugates etc.) may exist in, and be isolated in,isomeric forms, including tautomeric forms, cis- or trans-isomers, aswell as optically active, racemic, or diastereomeric forms. It is to beunderstood that the present invention encompasses a compound of formulaI in any of the tautomeric forms or as a mixture thereof, or as amixture of diastereomers, as well as in the form of an individualdiastereomer, and that the present invention encompasses a compound offormula I as a mixture of enantiomers, as well as in the form of anindividual enantiomer, any of which mixtures or form possessesinhibitory properties against kinases including PI-3 kinase, it beingwell known in the art how to prepare or isolate particular forms and howto determine inhibitory properties against kinases by standard testsincluding those described herein below.

In addition, a compound of formula I (or salt, procompound or conjugatethereof, etc.) may exhibit polymorphism or may form a solvate with wateror an organic solvent. The present invention also encompasses any suchpolymorphic form, any solvate or any mixture thereof.

As mentioned above, the invention includes a pharmaceutically acceptablesalt of a compound defined by the above formula I. A basic compound ofthis invention possesses one or more functional groups sufficientlybasic to react with any of a number of inorganic and organic acidsaffording a physiologically acceptable counterion to form apharmaceutically acceptable salt. Acids commonly employed to formpharmaceutically acceptable acid addition salts are inorganic acids suchas hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suchpharmaceutically acceptable salts thus are the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butane-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like. Preferredpharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid, hydrobromic acid andsulfuric acid.

D.1. Synthesis of Compounds and Conjugates

The compounds of the present invention may be prepared by processesknown in the chemical art. Starting materials and intermediates used toprepare a compound of the invention are either commercially available orcan be prepared by one of ordinary skill in the art.

The compounds of the invention, or their pharmaceutically acceptablesalts, may have asymmetric carbon atoms or quaternized nitrogen atoms intheir structure. It will be appreciated that certain compounds ofFormula I (or salts, conjugates, etc.) may exist in, and be isolated in,isomeric forms, including tautomeric forms, cis- or trans-isomers, aswell as optically active, racemic, or diastereomeric forms. It is to beunderstood that the present invention encompasses a compound of formulaI in any of the tautomeric forms or as a mixture thereof: or as amixture of diastereomers, as well as in the form of an individualdiastereomer, and that the present invention encompasses a compound ofFormula I as a mixture of entantiomers, as well as in the form of anindividual enantiomer, any of which mixtures or form possessesinhibitory properties against kinases, for example PI-3 kinases. Thecompounds of the invention and their pharmaceutically acceptable saltsmay exist as single stereoisomers, racemates, and as mixtures ofenantiomers and diastereomers. The compounds may also exist as geometricisomers. All such single stereoisomers, racemates and mixtures thereof,and geometric isomers are intended to be within the scope of theinvention.

Methods for the preparation and/or separation and isolation of singlestereoisomers from racemic mixtures or non-racemic mixtures ofstereoisomers are well known in the art. For example, optically active(R)- and (S)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using other conventional techniques. For example,enantiomers (R- and S-isomers) may be resolved by formation ofdiastereoisomeric salts or complexes which may be separated, forexample, by crystallization via formation of diastereoisomericderivatives which may be separated, for example, by crystallization,selective reaction of one enantiomer with an enantiomer-specificreagent, for example enzymatic oxidation or reduction, followed byseparation of the modified and unmodified enantiomers: or gas-liquid orliquid chromatography in a chiral environment, for example on a chiralsupport, such as silica with a bound chiral ligand or in the presence ofa chiral solvent. It will be appreciated that where a desired enantiomeris converted into another chemical entity by one of the separationprocedures described above, a further step may be required to liberatethe desired enantiomeric form. Alternatively, a specific enantiomer maybe synthesized by asymmetric synthesis using optically active reagents,substrates, catalysts or solvents or by converting on enantiomer to theother by asymmetric transformation. For a mixture of enantiomers,enriched in a particular enantiomer, the major component enantiomer maybe further enriched (with concomitant loss in yield) byrecrystallization.

A compound of Formula I in which R1 and R4 are hydrogen and R2 and R3have the meanings defined hereinabove may be synthesized as illustratedby Scheme I.

Another route to make a compound of Formula I in which R1 and R4 arehydrogen and R2 and R3 have the meanings defined hereinabove may besynthesized as illustrated by Scheme II.

Another route to make a compound of Formula I in which R1 and R4 arehydrogen and R2 and R3 have the meanings defined herein above may besynthesized as illustrated by Scheme III.

Another route to make a compound of Formula I in which R1 and R4 arehydrogen and R2 and R3 have the meanings defined hereinabove may besynthesized as illustrated by Scheme IV.

If methyl 4-bromo-3-hydroxythiophene-2-carboxylate is reacted with1-morpholinoethanone in Scheme I compounds of Formula V are produced:

A compound of Formula I-VII wherein a substituent of R1 (R3 for FormulasV-VII) further comprises a bone-directing group such as, for example,amino phosphonic acid, bisphosphonate, or the like, can be made byprocedures known in the art including, for example, the proceduresdisclosed in T. J. Houghton et al. J. Med. Chem. 2008, 51, 6955-6969,the entire contents of which is herein incorporated by reference.

D.2. Synthesis of Conjugates

Conjugates of the invention can be made, for example, by the proceduresdisclosed in U.S. Pat. Nos. 6,949,537 and 7,396,828, the entire contentsof which are herein incorporated by reference.

E. Formulations

As an additional aspect of the invention there is provided apharmaceutical formulation or composition comprising in association witha pharmaceutically acceptable carrier, diluent or excipient a compoundof the invention, e.g. Formula I (or a pharmaceutically acceptable saltor procompound or conjugate thereof) as provided in any of thedescriptions herein. Compositions of the present invention may be in theform of tablets or lozenges formulated in a conventional manner. Forexample, tablets and capsules for oral administration may containconventional excipients including, but not limited to, binding agents,fillers, lubricants, disintegrants and wetting agents. Binding agentsinclude, but are not limited to, syrup, acacia, gelatin, sorbitol,tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillersinclude, but are not limited to, lactose, sugar, microcrystallinecellulose, maizestarch, calcium phosphate, and sorbitol. Lubricantsinclude, but are not limited to, magnesium stearate, stearic acid, talc,polyethylene glycol, and silica. Disintegrants include, but are notlimited to, potato starch and sodium starch glycollate. Wetting agentsinclude, but are not limited to, sodium lauryl sulfate). Tablets may becoated according to methods well known in the art.

Compositions of the present invention may also be liquid formulationsincluding, but not limited to, aqueous or oily suspensions, solutions,emulsions, syrups, and elixirs. The compositions may also be formulatedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may contain additives including,but not limited to, suspending agents, emulsifying agents, nonaqueousvehicles and preservatives. Suspending agent include, but are notlimited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup,gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel, and hydrogenated edible fats. Emulsifying agents include,but are not limited to, lecithin, sorbitan monooleate, and acacia.Nonaqueous vehicles include, but are not limited to, edible oils, almondoil, fractionated coconut oil, oily esters, propylene glycol, and ethylalcohol. Preservatives include, but are not limited to, methyl or propylp-hydroxybenzoate and sorbic acid.

Compositions of the present invention may also be formulated assuppositories, which may contain suppository bases including, but notlimited to, cocoa butter or glycerides. Compositions of the presentinvention may also be formulated for inhalation, which may be in a formincluding, but not limited to, a solution, suspension, or emulsion thatmay be administered as a dry powder or in the form of an aerosol using apropellant, such as dichlorodifluoromethane or trichlorofluoromethane.Compositions of the present invention may also be formulated transdermalformulations comprising aqueous or nonaqueous vehicles including, butnot limited to, creams, ointments, lotions, pastes, medicated plaster,patch, or membrane.

Compositions of the present invention may also be formulated forparenteral administration including, but not limited to, by injection orcontinuous infusion. Formulations for injection may be in the form ofsuspensions, solutions, or emulsions in oil) or aqueous vehicles, andmay contain formulation agents including, but not limited to,suspending, stabilizing, and dispersing agents. The composition may alsobe provided in a powder form for reconstitution with a suitable vehicleincluding, but not limited to, sterile, pyrogen-free water.

Compositions of the present invention may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The compositions may be formulated withsuitable polymeric or hydrophobic materials (as an emulsion in anacceptable oil, for example), ion exchange resins, or as sparinglysoluble derivatives (as a sparingly soluble salt, for example).

Compositions of the present invention may also be formulated as aliposome preparation. The liposome preparation can comprise liposomeswhich penetrate the cells of interest or the stratum corneum, and fusewith the cell membrane, resulting in delivery of the contents of theliposome into the cell. For example, liposomes such as those describedin U.S. Pat. No. 5,077,211 of Yarosh, U.S. Pat. No. 4,621,023 ofRedziniak et al. or U.S. Pat. No. 4,508,703 of Redziniak et al. can beused. Other suitable formulations can employ niosomes. Niosomes arelipid vesicles similar to liposomes, with membranes consisting largelyof non-ionic lipids, some forms of which are effective for transportingcompounds across the stratum corneum.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way. The phrase“active ingredient” refers herein to a compound according to formula Ior a pharmaceutically acceptable salt, procompound, conjugate, orsolvate thereof.

Formulation 1: Tablet Containing the Following Components:

Ingredient Amount (mg/tablet) Active ingredient 250 Dried starch 200Magnesium stearate 10 Total 460 mg

Formulation 2: Capsules Containing the Following Components:

Ingredient Amount (mg/tablet) Active ingredient 60 Dried starch 44Magnesium stearate 1.5 Microcrystalline cellulose 44 Total 150 mg

Parenteral dosage forms for administration to patients by various routesincluding, but not limited to, subcutaneous, intravenous (includingbolus injection), intramuscular, and intra-arterial are alsocontemplated by the present invention. Parenteral dosage forms arepreferably sterile or capable of being sterilized prior toadministration to a patient. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection, andemulsions. Suitable vehicles that can be used to provide parenteraldosage forms of the invention are well known to those skilled in theart. Examples include, but are not limited to: Water for Injection USP,aqueous vehicles such as, but not limited to, Sodium Chloride Injection.Ringer's Injection, Dextrose Injection. Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

An example parenteral composition of the invention would be intended fordilution with aqueous solution(s) comprising for example 5% DextroseInjection, USP, or 0.9% Sodium Chloride Injection, USP, prior toadministration to a patient, and is an aqueous solution that comprisesirinotecan, sorbitol NF powder, and lactic acid. USP, and has a pH offrom about 3.0 to about 3.8.

F. Therapeutic Use

The present invention also encompasses medical use of compounds of thepresent invention including methods of treatment of a patient sufferingfrom a condition or disease associated with aberrant kinase activityincluding PI-3 kinase. In one aspect, kinase activity may be abnormal,excessive, or constitutively active in a patient in need of suchtreatment. The present invention also relates to a method for treatinginflammatory disease comprising administering to a patient in needthereof a therapeutically effective amount of a compound of the presentinvention. Exemplary, but non-exclusive diseases and adverse healthconditions attributable to kinase activity, in particular inappropriatePI-3 kinase signaling activity, have been disclosed in the art, forexample U.S. 2002/0150954A1; U.S. Pat. No. 5,504,103; U.S. Pat. No.6,518,277B1; U.S. Pat. No. 6,403,588; U.S. Pat. No. 6,482,623: U.S. Pat.No. 6,518,277; U.S. Pat. No. 6,667,300. U.S.20030216389;U.S.20030195211; U.S.20020037276 and U.S. Pat. No. 5,703,075 thecontents of which are herein incorporated by reference.

The compounds of the invention can have utility in the treatment of CNSdisorders, including schizophrenia, episodic paroxysmal anxiety (EPA)disorders such as obsessive compulsive disorder (OCD), post traumaticstress disorder (PTSD), phobia and panic, major depressive disorder,bipolar disorder, Parkinson's disease, general anxiety disorder, autism,delirium, multiple sclerosis, Alzheimer disease/dementia and otherneurodegenerative diseases, severe mental retardation, dyskinesias.Huntington's disease. Tourett's syndrome, tics, tremor, dystonia,spasms, anorexia, bulimia, stroke, addiction/dependency/craving, sleepdisorder, epilepsy, migraine; and attention deficit/hyperactivitydisorder (ADHD).

In another aspect, the present invention provides a method for treatingAlzheimer's Disease comprising administering to a patient in needthereof a therapeutically effective amount of a compound of the presentinvention. It has been reported that increasing PIP2 concentrations by,for example, inhibiting PI-3 kinase decreases levels of neurotoxinsassociated with Alzheimer's Disease. (US 2008/0312187; incorporatedherein by reference). 0118) In another aspect, the present inventionprovides a method for enhancing the chemosensitivity of tumor cellscomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of the present invention.

In another aspect, the present invention provides a method for enhancingthe radiosensitivity of tumor cells comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof the present invention.

In another aspect, the present invention provides a method forinhibiting or reducing tumor growth comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof the present invention.

In another aspect, the present invention provides a method for inducingoxidative stress in tumor cells comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of thepresent invention.

In another aspect, the present invention provides a method forinhibiting or reducing tumor growth by inhibiting cancer stem cellgrowth and/or proliferation comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of thepresent invention.

In another aspect, the present invention provides a method forinhibiting tumor induced angiogenesis comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof the present invention.

Further, the present invention provides a method for inhibitingangiogenesis associated with non-cancer diseases comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound of the present invention.

In yet another aspect, the present invention provides a therapeuticmethod for increasing apoptosis in cancer cells and cancerous tumorscomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of the present invention.

The present invention also provides a method of treating cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of the present invention.

A variety of cancers may be treated according to the present inventionincluding, but not limited to: carcinoma of the bladder (includingaccelerated and metastatic bladder cancer), breast, colon (includingcolorectal cancer), kidney, liver, lung (including small and non-smallcell lung cancer and lung adenocarcinoma), ovary, prostate, testes,genitourinary tract, lymphatic system, rectum, larynx, pancreas(including exocrine pancreatic carcinoma), esophagus, stomach, gallbladder, cervix, thyroid, and skin (including squamous cell carcinoma);hematopoietic tumors of lymphoid lineage including leukemia, acutelymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma,T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy celllymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietictumors of myeloid lineage including acute and chronic myelogenousleukemias, myelodysplastic syndrome, myeloid leukemia, and promyelocyticleukemia; tumors of the central and peripheral nervous system includingastrocytoma, neuroblastoma, glioma, and schwannomas: tumors ofmesenchymal origin including fibrosarcoma, rhabdomyosarcoma, andosteosarcoma; and other tumors including melanoma, xenodermapigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, andteratocarcinoma. The methods of the invention may also be used to treataccelerated or metastatic cancers of the bladder, pancreatic cancer,prostate cancer, non-small cell lung cancer, colorectal cancer, andbreast cancer.

A compound of the invention may be administered simultaneously ormetronomically with other anti-cancer treatments such as chemotherapyand radiation therapy. The term “simultaneous” or “simultaneously” asused herein, means that the other anti-cancer treatment and the compoundof the present invention are administered within 48 hours, preferably 24hours, more preferably 12 hours, yet more preferably 6 hours, and mostpreferably 3 hours or less, of each other. The term “metronomically” asused herein means the administration of the compounds at times differentfrom the chemotherapy and at a certain frequency relative to repeatadministration and/or the chemotherapy regimen.

The chemotherapy treatment may comprise administration of a cytotoxicagent or cytostatic agent, or combination thereof. Cytotoxic agentsprevent cancer cells from multiplying by: (1) interfering with thecell's ability to replicate DNA and (2) inducing cell death and/orapoptosis in the cancer cells. Cytostatic agents act via modulating,interfering or inhibiting the processes of cellular signal transductionwhich regulate cell proliferation and sometimes at low continuouslevels.

Classes of compounds that may be used as cytotoxic agents include butare not limited to the following: alkylating agents (including, withoutlimitation, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas and triazenes): uracil mustard, chlormethine,cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil,pipobroman, triethylene-melamine, triethylenethiophosphoramine,busulfan, carmustine, lomustine, streptozocin, dacarbazine, andtemozolomide; antimetabolites (including, without limitation, folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors): methotrexate, 5-fluorouracil floxuridine, cytarabine,6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine,and gemcitabine: natural products and their derivatives (for example,vinca alkaloids, antitumor antibiotics, enzymes, lymphokines andepipodophyllotoxins): vinblastine, vincristine, vindesine, bleomycin,dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c,paclitaxel (paclitaxel is commercially available as Taxol®),mithramycin, deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons(preferably IFN-.alpha.), etoposide, and teniposide. Other proliferativecytotoxic agents are navelbene, CPT-11, anastrazole, tetrazole,capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

Microtubule affecting agents interfere with cellular mitosis and arewell known in the art for their cytotoxic activity. Microtubuleaffecting agents useful in the invention include, but are not limitedto, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives (e.g., NSC608832), thiocolchicine NSC 361792), trityl cysteine (NSC 83265),vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574),natural and synthetic epothilones including but not limited toepothilone A, epothilone B, and discodermolide (see Service, (1996)Science, 274:2009) estramustine, nocodazole, MAP4, and the like.Examples of such agents are also described in J. C. Bulinski et al. J.Cell Sci. 1997, 110, 3055-3064: D. Panda et al. Proc. Natl. Acad. Sci.USA 1997, 94, 10560-10564; P. F. Mühradt et al. Cancer Res. 1997, 57,3344-3346: K. C. Nicolaou et al. Nature 1997, 387, 268-272; R. J.Vasquez et al Mol. Biol. Cell. 1997, 8, 973-985; and D. Panda et al. J.Biol. Chem. 1996, 271, 29807-29812.

Other suitable cytotoxic agents include but are not limited toepidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;procarbazine; mitoxantrone; platinum coordination complexes such ascis-platin and carboplatin; biological response modifiers: growthinhibitors: antihormonal therapeutic agents; leucovorin; tegafur; andhaematopoietic growth factors.

Cytostatic agents that may be used according to the invention include,but are not limited to, hormones and steroids (including syntheticanalogs): 17 alpha-ethinylestradiol, diethylstilbestrol, testosterone,prednisone, fluoxymesterone, dromostanolone propionate, testolactone,megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone,triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide,estramustine, medroxyprogesteroneacetate, leuprolide, flutamide,toremifene, zoladex. Other cytostatic agents are antiangiogenics such asmatrix metalloproteinase inhibitors, and other VEGF inhibitors, such asanti-VEGF antibodies and small molecules such as ZD6474 and SU6668 arealso included. Anti-Her2 antibodies from Genentech may also be utilized.A suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Alsoincluded are Imclone antibody C225 immunospecific for the EGFR, and srcinhibitors. Also suitable for use as a cytostatic agent is Casodexlk;(bicalutanude, Astra Zeneca) which renders androgen-dependent carcinomasnon-proliferative. Yet another example of a cytostatic agent is theantiestrogen Tamoxifen®, which inhibits the proliferation or growth ofestrogen dependent breast cancer. Inhibitors of the transduction ofcellular proliferative signals are cytostatic agents. Representativeexamples include but are not limited to epidermal growth factorinhibitors, Her-2 inhibitors, MEK-I kinase inhibitors, MAPK kinaseinhibitors, PI3 inhibitors, Src kinase inhibitors, and PDGF inhibitors.

The present invention also encompasses a method for treatingpancreatitis comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the present invention.As discussed in I. Gukovsky et al. Gastroenterology 2004, 126, 554-566,inhibition of PI-3 kinase may prevent pancreatitis.

The present invention also encompasses a method for treating ulcerscomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of the present invention. The presentinvention also encompasses a method for treating gastric cancer, such asstomach cancer, comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the present invention.As discussed in Bacon et al., Digestive Disease Week Abstracts andItinerary Planner, Vol. 2003, Abstract No. M921 (2003) and Rokulan etal., Digestive Disease Week Abstracts and Itinerary Planner. Vol. 2003.Abstract No. 354 (2003), PI-3 kinase is involved in the adhesion ofHelicobacter pylori to gastric cells.

The present invention also encompasses a method for treating age-relatedmacular degeneration (AMD) comprising administering to a patient in needthereof a therapeutically effective amount of a compound of the presentinvention. As discussed in Retina, Feb. 18, 2004, inhibition of VEGFinhibits blood vessel overgrowth associated with AMD. The compounds ofthe present invention may treat AMD by inhibiting angiogenesis.

The present invention also encompasses a method for treating conditionsassociated with a mutant PTEN comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of thepresent invention. PTEN is a tumor suppressor gene located on chromosome10q23, in which mutations have been identified in patients with Cowdendisease. As discussed in A. Vega et al. J. Invest. Dermatol. 2003, 121,1356-1359, mutations in PTEN have reduced ability to inhibit theactivation of the proto-oncogene AKT. Inhibitors of PI-3 kinase mayinhibit phosphorylation of AKT, thereby reducing the deleterious effectof mutant PTEN.

G. Administration and Dosage

A compound or composition of the present invention may be administeredin any manner including but not limited to orally, parenterally,sublingually, transdermally, rectally, transmucosally, topically,pulmonarily, nasally, or bucally. Parenteral administration includes butis not limited to intravenous, intraarterial, intraperitoneal,subcutaneous, intramuscular, intrathecal, and intraarticular. Thecompound or compositions of the invention may also be administered viaslow controlled i.v. infusion or by release from an implant device.

A therapeutically effective amount of a compound of the inventionrequired for use in therapy varies with the nature of the conditionbeing treated, the length of treatment time desired, the age and thecondition of the patient, and is ultimately determined by the attendingphysician. In general, however, doses employed for adult human treatmenttypically are in the range of 0.001 mg/kg to about 200 mg/kg per day.The dose may be about 1 μg/kg to about 100 μg/kg per day. The desireddose may be conveniently administered in a single dose, or as multipledoses administered at appropriate intervals, for example as two, three,four or more sub-doses per day. Multiple doses often are desired, orrequired.

A number of factors may lead to the compounds of the present inventionbeing administered over a wide range of dosages. When given incombination with other therapeutic agents, the dosage of the compoundsof the present invention may be given at relatively lower dosages. Inaddition, the use of targeting agents on a conjugate of the invention isexpected to lower the effective dosage required for treatment. As aresult, the daily dosage of a compound of the present invention may befrom about 1 ng/kg to about 100 mg/kg. The dosage of a compound of thepresent invention may be at any dosage including, but not limited to,about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 125 μg/kg, 150μg/kg, 175 μg/kg, 200 μg/kg. 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg,325 g/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg,650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg,975 μg/kg, 1 mg/kg, 5 mg/kg. 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80mg/kg, 90 mg/kg, or 100 mg/kg.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples. The examples are merely illustrative and do notlimit the scope of the invention in any way.

H. Preparation of Compounds Synthesis of1-(4-bromo-3-hydroxythiophen-2-yl)-3-morpholinopropane-1,3-dione

An oven-dried 100 mL round-bottom flask was charged with a magneticstirring bar, tetrahydrofuran (5 mL) and lithium diisopropylamide (2.0Min hexanes, 6.7 mL, 13.5 mmol). The reaction mixture was magneticallystirred and cooled to 0° C. under argon gas. A solution ofN-acetylmorpholine (6.75 mmol) in tetrahydrofuran (1 mL) was added, andthe reaction was stirred at 0° C. for 1 hour. A solution of methyl4-bromo-3-hydroxythiophene-2-carboxylate (4.22 mmol) in tetrahydrofuran(2 mL) was added dropwise over 5 minutes and the reaction was allowed towarm to room temperature overnight. The reaction was quenched by theaddition of aqueous 10% hydrochloric acid solution (20 mL). Theresulting solution was transferred to a separatory funnel, diluted withwater (80 mL), and extracted three times with dichloromethane (100 mL).The organic layers were combined, dried over magnesium sulfate,filtered, and concentrated in vacuo to give1-(4-bromo-3-hydroxythiophen-2-yl)-3-morpholinopropane-1,3-dione (1.95g, 5.83 mmol, 138% yield) as a brown solid.

Synthesis of 3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one

An oven-dried 100 mL round-bottom flask was charged with a magneticstirring bar,1-(4-bromo-3-hydroxythiophen-2-yl)-3-morpholinopropane-1,3-dione (1.95g, 5.83 mmol) and dissolved in dichloromethane (30 mL) under magneticstirring. Trifluoromethanesulfonic anhydride (2.45 mL, 14.6 mmol) wasadded portionwise over 2 minutes, and the reaction was stirred at roomtemperature. After stirring overnight, the reaction was concentrated invacuo and redissolved in methanol (10 mL). After stirring for 4 hours,the reaction was concentrated in vacuo and diluted with aqueous 5%sodium bicarbonate solution (100 mL). The reaction was transferred to aseparatory funnel and extracted three times with dichloromethane (100mL). The organic layers were combined, dried over magnesium sulfate,filtered, and concentrated in vacuo to give3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (1.52 g, 4.81mmol, 82%) as a brown solid.

Suzuki Coupling Reaction Procedure A Synthesis of5-morpholino-3-phenyl-7H-thieno[3,2-b]pyran-7-one (6)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (50 mg, 0.16mmol), phenylboronic acid (29 mg, 0.24 mmol), cesium carbonate (103 mg,0.32 mmol), tetrakis(triphenylphosphine)palladium(0) (9 mg, 0.008 mmol),and dimethoxyethane (1 mL). The reaction mixture was magneticallystirred and heated via microwave irradiation for 15 minutes at 130° C.Upon cooling to room temperature, the reaction was concentrated in vacuoand purified using high-pressure liquid chromatography to give5-morpholino-3-phenyl-7H-thieno[3,2-b]pyran-7-one (6) (10 mg, 3.2 μmol)as a white solid.

Suzuki Coupling Reaction Procedure B Synthesis of5-morpholino-3-(pyridin-3-yl)-7H-thieno[3,2-b]pyran-7-one (3)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (50 mg, 0.16mmol), 3-pyridylboronic acid (29 mg, 0.24 mmol), cesium carbonate (103mg, 0.32 mmol),dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethane adduct (9 mg, 0.008 mmol), and dimethoxyethane (1 mL).The reaction mixture was magnetically stirred and heated via microwaveirradiation for 15 minutes at 180° C. Upon cooling to room temperature,the reaction was concentrated in vacuo and purified using high-pressureliquid chromatography to give5-morpholino-3-(pyridin-3-yl)-7H-thieno[3,2-b]pyran-7-one (3) (6 mg, 1.9μmol) as a white solid.

Sonogashira Coupling Reaction Procedure C5-morpholino-3-(phenylethynyl)-7H-thieno[3,2-b]pyran-7-one (31)

A microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (50 mg, 158.1μmol), phenylacetylene (32.0 mg, 316.2 μmol),trans-dichlorobis(triphenylphosphine)palladium (11) (5.6 mg, 7.9 tμmol),copper (1) iodide (1.6 mg, 7.9 μmol) and diisopropylamine (1.0 mL). Themixture was magnetically stirred and heated via microwave irradiation to100° C. for 20 minutes. The mixture was cooled to room temperature, andthen concentrated in vacuo resulting in a brown solid. The solid wasdissolved in methanol (1.0 mL) and loaded onto Isolute functionalizedsilica column (PE-AX/SCX-2) The column was washed with methanol and 7Nmethanolic ammonia. The methanolic ammonia fractions were combined andconcentrated in vacuo. The resulting solid was purified usingpreparative high-pressure liquid chromatography to give5-morpholino-3-(phenylethynyl)-7H-thieno[3,2-b]pyran-7-one (31) (24.0mg, 71.1 μmol) as an orange solid.

Aminocarbonylation Reaction Procedure D5-morpholino-7-oxo-N-phenyl-7H-thieno[3,2-b]pyran-3-carboxamide (51)

A 2 mL microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (100 mg, 316μmol), molybdenumhexacarbonyl (42 mg, 160 μmol),trans-di(μ-acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium (11)(4.6 mg, 4.9 μmol), tri-tert-butylphosphonium tetrafluoroborate (2.8 mg,12.0 μmol), tetrahydrofuran (500 μL), aniline (45 μL, 483 μmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (32 μL, 210 μmol). The vial wasimmediately sealed, magnetically stirred and heated via microwaveirradiation to 125° C. for 6 minutes. The mixture was cooled to roomtemperature, and loaded onto Isolute functionalized silica column(PE-AX/SCX-2). The column was washed with methanol and the product waseluted with 7N methanolic ammonia. The methanolic ammonia fractions werecombined and concentrated in vacuo. The resulting solid was purifiedusing preparative high-pressure liquid chromatography to give5-morpholino-7-oxo-N-phenyl-7H-thieno[3,2-b]pyran-3-carboxamide (51),(3.88 mg, 10.9 μmol) as a yellow solid.

Reverse Suzuki Coupling Reaction Procedure E Synthesis of5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-ylboronic acid

A 20 mL microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (1.20 g: 3.80mmol), 1,3-bis(diphenylphosphino)propane (156 mg, 0.38 mmol),1,3-bis(diphenylphosphino)propane nickel(II) chloride (205 mg, 0.38mmol), dioxane (8 mL), diisopropylethylamine (1.98 mL, 11.39 mmol), and4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.10 mL, 7.59 mmol). Thereaction mixture magnetically stirred and heated via microwaveirradiation for 15 minutes at 160° C. Upon cooling to room temperature,the reaction was transferred to a separatory funnel, diluted withdichloromethane (100 mL), and washed three times with saturated aqueousammonium chloride solution (100 mL). The dichloromethane layer was driedover magnesium sulfate, filtered, and concentrated in vacuo. Theresulting brown foam was stirred in aqueous hydrochloric acid (1.0M, 100mL) for 30 minutes, frozen, and lyophilized to give5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-ylboronic acid (69) (1.51 g,4.15 mmol) as a brown solid.

Synthesis of methyl3-hydroxy-4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)thiophene-2-carboxylate(21)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-ylboronic acid (69) (57 mg,0.16 mmol), cesium carbonate (102 mg, 0.31 mmol), methyl4-bromo-3-hydroxythiophene-2-carboxylate (56 mg, 0.24 mmol),tetrakis(triphenylpbosphine)palladium(0) (9 mg, 0.08 mmol), anddimethoxyethane (0.5 mL). The reaction mixture was magnetically stirredand heated via microwave irradiation for 15 minutes at 140° C. Uponcooling to room temperature, the reaction was concentrated in vacuo andpurified using high-pressure liquid chromatography to give methyl3-hydroxy-4-(5-morpholino-7-oxo-7H-thieno[32-b]pyran-3-yl)thiophene-2-carboxylate(21).

Acylation Reaction Procedure F Synthesis ofN-(4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)phenyl)nicotinamide(30)

A 40 mL vial was charged with a magnetic stirring bar,3-(4-aminophenyl)-5-morpholino-7H-thieno[3,2-b]pyran-7-one (56) (200 mg,609 μmol) and pyridine (5 mL). The mixture was magnetically stirred andcooled to 0° C. in an ice bath. Isonicotinoyl chloride hydrochloric acid(669 μmol) was added and the reaction was stirred at room temperaturefor 30 minutes. The reaction was diluted with dichloromethane (15 mL)and washed three times with saturated aqueous sodium bicarbonatesolution (15 mL). The dichloromethane layer was dried over magnesiumsulfate, filtered, and concentrated in vacuo to giveN-(4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)phenyl)nicotinamide(30) (94 mg, 217 μmol) as a yellow solid.

Aminocarbonylation Reaction Procedure G Synthesis ofN-benzyl-4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)benzamide (50)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,3-(4-chlorophenyl)-5-morpholino-7H-thieno[3,2-b]pyran-7-one (17) (80 mg,230 μmol),trans-di(μ-acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium (II)(6 mg, 7 μmol), tri-tert-butylphosphonium tetrafluoroborate (4 mg, 14μmol), molybdenumhexacarbonm (61 mg, 230 μmol), tetrahydrofuran (500μL), benzyl amine (75 μL, 69) μmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (103 μL, 690 μmol). The reactionmixture was magnetically stirred and heated via microwave irradiationfor 15 minutes at 170° C. Upon cooling to room temperature, the reactionwas concentrated in vacuo and purified using high-pressure liquidchromatography to giveN-benzyl-4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)benzamide(50).

Synthesis of Pyran Analogs Procedure H Synthesis of1-(2-Hydroxybiphenyl-3-yl)-3-(tetrahydro-2H-pyran-4-yl)propane-1,3-dione

An oven-dried 100 mL round-bottom flask was charged with a magneticstirring bar, tetrahydrofuran (10 mL) and lithium diisopropylamide (2.0Min hexanes, 6.9 mL, 13.8 mmol). The mixture was magnetically stirred andcooled to 0° C. under argon gas, 1-(2-Hydroxybiphenyl-3-yl)ethanone(0.92 g, 4.34 mmol) was added, and the reaction was stirred at 0° C. for1 hour. A solution of methyl tetrahydro-2H-pyran-4-carboxylate (1.0 g,6.94 mmol) in tetrahydrofuran (2 mL) was added dropwise over 5 minutesand the reaction was allowed to warm to room temperature overnight. Thereaction was quenched by the addition of aqueous 10% hydrochloric acidsolution (20 mL) until pH=3. The resulting solution was transferred to aseparatory funnel, diluted with water (80 mL), and extracted three timeswith dichloromethane (100 mL). The organic layers were combined, driedover magnesium sulfate, filtered, and concentrated in vacuo to givecrude1-(2-hydroxybiphenyl-3-yl)-3-(tetrahydro-2H-pyran-4-yl)propane-1,3-dioneas a brown oil. The crude product was used as is without furtherpurification.

Synthesis of 8-Phenyl-2-(tetrahydro-2H-pyran-4-yl)-4H-chromen-4-one (67)

A 5 mL microwave vial was charged with a magnetic stirring bar, crude1-(2-hydroxybiphenyl-3-yl)-3-(tetrahydro-2H-pyran-4-yl)propane-1,3-dione(320 mg, 1 mmol), ethanol (3 mL), and copper (II) chloride (13 mg, 0.1mmol). The reaction vial was sealed, the reaction mixture magneticallystirred and heated via microwave irradiation to 140° C. for 10 minutes.The resulting solution was concentrated in vacuo and purified byhigh-pressure liquid chromatography to give8-phenyl-2-(tetrahydro-2H-pyran-4-yl)-4H-chromen-4-one (67).

Thioketone Synthesis Procedure I Synthesis of2-morpholino-8-phenyl-4H-chromene-4-thione (68)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,2-morpholino-8-phenyl-4H-chromen-4-one (LY294002) (150 mg, 488 μmol),Lawesson's reagent (118 mg, 293 μmol), and toluene (2 mL). The reactionmixture was sealed, and the reaction mixture was magnetically stirredand heated via microwave irradiation to 130° C. for 20 minutes. Thefinal mixture was poured onto water (approximately 30 mL), extractedwith dichloromethane (3×5 mL), the combined extracts dried overanhydrous magnesium sulfate, filtered and concentrated to dryness.Purification via column chromatography (silica gel: hexanes/ethylacetate (1:1), then 100% ethyl acetate) afforded pure2-morpholino-8-phenyl-4H-chromene-4-thione (68) (128 mg, 81% yield).

Carboxylic Acid Synthesis Via Carbonyl Insertion Procedure J Synthesisof 5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-carboxylic acid (70)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (50 mg, 158μmol), molybdenum hexacarbonyl (42 mg, 158 μmol),trans-di(t-acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium (II)(2.0 mg, 2.3 μmol), tri-tert-butylphosphonium tetrafluoroborate (1.4 mg,4.7 μmol), dimethyl sulfoxide (0.5 mL), water (2.8 μL, 158 μmol), and1,8-diazabicyclo[5.4.0]undec-7-ene (16 μL. 106 μmol). The reactionmixture was sealed, and the reaction mixture was magnetically stirredand heated via microwave irradiation to 125° C. for 6 minutes to give5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-carboxylic acid (70).

Thiophene Amination Reaction Procedure K Synthesis of5-morpholino-3-(piperidin-1-yl)-7H-thieno[3,2-b]pyran-7-one (71)

A 0.5 mL microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (50 mg, 158gμmol), cesium carbonate (103 mg, 316 μmol), copper (I) iodide (3 mg, 31μmol), ethyl 2-oxocyclohexanecarboxylate (10 μL, 63 μmol), andpiperidine (200 μL). The reaction mixture was sealed, and the reactionmixture was magnetically stirred and heated via microwave irradiation to150° C. for 15 minutes to give5-morpholino-3-(piperidin-1-yl)-7H-thieno[3,2-b]pyran-7-one (71).

Phenyl Amination Reaction Procedure L Synthesis of5-morpholino-3-(4-morpholinophenyl)-7H-thieno[3,2-b]pyran-7-one (72)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,3-(4-chlorophenyl)-5-morpholino-7H-thieno[3,2-b]pyran-7-one (17) (80 mg,230 μmol), sodium tert-butoxide (31 mg, 322 μmol),tris(dibenzylideneacetone)dipalladium(0) (3 mg, 3.5 μmol),2-dicyclohexylphosphino-2′.4′.6′-triisopropylbiphenyl (8.8 mg, 21 μmol),dimethoxyethane (0.5 mL), and morpholine (24 μL, 276 μmol). The reactionmixture was sealed, and the reaction mixture was magnetically stirredand heated via microwave irradiation to 150° C. for 15 minutes to give5-morpholino-3-(4-morpholinophenyl)-7H-thieno[3,2-b]pyran-7-one (72).

Thiophene Carboxylic Ester Synthesis Via Carbonyl Insertion Procedure MSynthesis of phenyl5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-carboxylate (73)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (50 mg, 158μmol), phenol (22 mg, 237 μmol), molybdenum hexacarbonyl (42 mg, 158μmol), trans-di(l-acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium(11) (4 mg, 4.7 μmol), tri-tert-butylphosphonium tetrafluoroborate (2.8mg, 9.5 μmol), tetrahydrofuran (0.5 mL), and1,8-diazabicyclo[5.4.0]undec-7-ene (71 μL, 474 μmol). The reactionmixture was sealed, and the reaction mixture was magnetically stirredand heated via microwave irradiation to 125° C. for 6 minutes to givephenyl 5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-carboxylate (73).

Thiophene Ketone Synthesis Via Carbonyl Insertion Procedure N Synthesisof 3-acetyl-5-morpholino-7H-thieno[3,2-b]pyran-7-one (74)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (100 mg, 316μmol), dimethylformamide (0.5 mL), N,N-diisopropylethylamine (165 μL,949 μmol), butyl vinyl ether (124 μL, 949 μmol), anddichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethane adduct (10 mg, 12.6 μmol). The reaction mixture wassealed, and the reaction mixture was magnetically stirred and heated viamicrowave irradiation to 180° C. for 15 minutes. After cooling to roomtemperature, the reaction was charged with aqueous hydrochloric acid(1.0 M, 2 mL) and stirred at room temperature for 2 hours to give3-acetyl-5-morpholino-7H-thieno[3,2-b]pyran-7-one (74).

Phenyl Carboxylic Ester Synthesis Via Carbonyl Insertion Procedure OSynthesis of butyl3-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)benzoate (75)

A 2 mL microwave vial was charged with a magnetic stirring bar,3-(3-chlorophenyl)-5-morpholino-7H-thieno[3,2-b]pyran-7-one (85) (80 mg,230 μmol), imidazole (47 mg, 690 μmol), molybdenum hexacarbonyl (60 mg,230 umol),trans-di(μ-acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium (11)(6.5 mg, 6.9 μmol), tri-tert-butylphosphonium tetrafluoroborate (4.0 mg,13.9 μmol), tetrahydrofuran (500 μL), n-butanol (63 μL, 690 μmol), and1,8-diazabicyclo[5.4.0]undec-7-ene (103 μL, 690 μmol). The vial wasimmediately sealed, and the reaction mixture was magnetically stirredand heated via microwave irradiation to 170° C. for 15 minutes in amicrowave reactor to give butyl3-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)benzoate (75).

Selective Thiophene Carbon-Carbon Coupling Reaction Procedure PSynthesis of 3-bromo-5-morpholino-2-phenyl-7H-thieno[3,2-b]pyran-7-one(76)

A 2 mL conical microwave vial was charged with a magnetic stirring bar,3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (50 mg, 158μmol), phenyl iodide (27 mg, 132 mol), potassium fluoride (11.5 mg, 198μmol), silver nitrate (34 mg, 198 μmol), anhydrous dimethyl sulfoxide (1mL), and dichlorobis(triphenylphosphine)palladium (II) (4.6 mg, 6.6μmol). The reaction mixture was sealed, and the reaction mixture wasmagnetically stirred and heated under microwave irradiation to 120° C.for 15 minutes to give3-bromo-5-morpholino-2-phenyl-7H-thieno[3,2-b]pyran-7-one (76).

Bis(pinacolato)diboron-mediated Carbon-Carbon Coupling ReactionProcedure R Synthesis of 2-morpholinoethyl 4-iodobenzoate

A 8 mL vial was charged with 2-morpholinoethanol (200 μL, 1.63 mmol),dichloromethane (5 mL), polystyrene-linked diisopropylamine (4.90 mmol),and 4-iodobenzoyl chloride (479 mg, 1.80 mmol), and the reaction mixturewas shaken at room temperature. After 16 hours, the reaction mixture wascharged with polystyrene-linked tris(2-aminoethyl)-amine (1.63 mmol),and shaken at room temperature for 2 hours. The reaction was filtered,and the resin was washed with dichloromethane (3×10 mL), tetrahydrofuran(2×10 mL), and diethyl ether (2×10 mL). The combined filtrates wereconcentrated in vacuo to give 2-morpholinoethyl 4-iodobenzoate (434 mg,1.19 mmol) as a white solid.

Synthesis of 2-morpholinoethyl4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)benzoate (77)

A 2 mL microwave vial was charged with a magnetic stirring bar,2-morpholinoethyl 4-iodobenzoate (90 mg, 249 μmol),3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (103) (79 mg, 249μmol), bis(pinacolato)diboron (70 mg, 274 μmol), cesium carbonate (325mg, 997 μmol),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethane adduct (6 mg, 7 μmol), and 1,2-dimethoxyethane (1 mL).The reaction mixture was sealed, and the reaction mixture wasmagnetically stirred and heated under microwave irradiation to 140° C.for 15 minutes to give 2-morpholinoethyl4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)benzoate (77).

Conjugates: Chloro Pyrillum Procedure S Synthesis of4-chloro-2-morpholino-8-phenylchromenylium chloride (78)

A solution of 2-morpholino-8-phenyl-4H-chromen-4-one (LY294002) (1.50 g,4.93 mmol) and dichloromethyl methyl ether (15 mL) was stirred andheated to 80° C. for 2 hours under argon. The mixture was cooled to roomtemperature, and then concentrated in vacuo to give4-chloro-2-morpholino-8-phenylchromenylium chloride (78) (2.06 g, 5.73mmol).

Conjugates: Pyrillum Ether Procedure T Synthesis of4-(3-hydroxypropoxy)-2-morpholino-8-phenylchromenylium chloride (79)

A 2 mL microwave vial was charged with a magnetic stirring bar,4-chloro-2-morpholino-8-phenylchromenylium chloride (78) (50 mg, 138μmol), 1,3-propanediol (I mL), and N,N-diisopropylethylamine (48 μL, 276μmol). The vial was sealed, and the reaction mixture was magneticallystirred and heated under microwave irradiation to 120° C. for 10 minutesin a microwave reactor to give4-(3-hydroxypropoxy)-2-morpholino-8-phenylchromenylium chloride (79).

Conjugates: Pyrillum Ether Procedure U Synthesis of4-ethoxy-2-morpholino-8-phenylchromenylium chloride (80)

A vial was charged with a magnetic stirring bar,4-chloro-2-morpholino-8-phenylchromenylium chloride (78) (50 mg, 138μmol), chloroform (1.0 mL), and a solution of sodium ethoxide in ethanol(21%, 108 IμL, 290 μmol). The mixture was magnetically stirred at roomtemperature for 1 hour to give4-ethoxy-2-morpholino-8-phenylchromenylium chloride (80).

Conjugates: Pyrillum Ether Procedure V Synthesis of7-((4-tert-Butoxy-4-oxobutanoyloxy)methoxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-iumiodide (81)

A 10 mL vial was charged with a magnetic stirring bar, tert-butylchloromethyl succinate (24 mg, 0.107 mmol), acetonitrile (2 mL),3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholino-7H-thieno[3,2-b]pyran-7-one(28) (20 mg, 0.054 mmol) and sodium iodide (12 mg, 0.081 mmol). Thereaction mixture was magnetically stirred for 18 hours at 70° C. to give7-((4-tert-butoxy-4-oxobutanoyloxy)methoxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-iumiodide (81).

Conjugates: Pyrillum Acid Chloride Procedure W Synthesis of7-((4-chloro-4-oxobutanoyloxy)methoxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-ium chloride (82)

A vial was charged with a magnetic stirring bar,7-((4-tert-butoxy-4-oxobutanoyloxy)methoxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-ium iodide (81)(1.02 g, 1.49 mmol), dichloromethane (4.7 mL) and hydrochloric acid (4 Min dioxane, 1.49 mL, 5.95 mmol). The solution was magnetically stirredat room temperature for 30 minutes. The reaction was charged withthionyl chloride (1.63 mL, 2.98 mmol) and stirred at room temperatureovernight. The reaction solution was concentrated in vacuo to give7-((4-chloro-4-oxobutanoyloxy)methoxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-iumchloride (82) (1.12 g, 2.01 mmol) as a crunchy yellow solid.

Conjugates: Pyrillum Protected RGDS Procedure X Synthesis of7-((8S,14S,17S)-14-(2-tert-butoxy-2-oxoethyl)-17-(tert-butoxymethyl)-20,20-dimethyl-3,6,9,12,15,18-hexaoxo-8-(3-(3-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfonyl)guanidino)propyl)-2,19-dioxa-7,10,13,16-tetraazahenicosyloxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-iumchloride (83)

A vial was charged with a magnetic stirring bar,H-Arg(Pbf)-Gly-Asp(O^(t)Bu)-Ser(^(t)Bu)—O^(t)Bu (854 mg, 1.00 mmol),dichloromethane (10 mL), and N-methylmorpholine (340 μL, 3.09 mmol). Themixture was cooled to 0° C. under magnetic stirring and then chargedwith7-((4-chloro-4-oxobutanoyloxy)methoxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-iumchloride (82) (500 mg, 0.77 mmol). The reaction solution was allowed towarm to room temperature and stirred for 1 hour. The reaction solutionwas diluted with dichloromethane (50 mL), washed three times with diluteaqueous citric acid (0.1 M, 50 mL), washed three times with diluteaqueous sodium bicarbonate solution (5%, 50 mL), washed twice with brinesolution, dried over magnesium sulfate, filtered, and concentrated invacuo to give7-((8S,14S,17S)-14-(2-tert-butoxy-2-oxoethyl)-17-(tert-butoxymethyl)-20,20-dimethyl-3,6,9,12,15,18-hexaoxo-8-(3-(3-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfonyl)guanidino)propyl)-2,19-dioxa-7,10,13,16-tetraazahenicosyloxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-iumchloride (83) (1.05 g, 0.72 mmol) as a yellow solid.

Conjugates: Pyrillum Unprotected RGDS Procedure Y Synthesis of7-((8S,14S,17S)-17-carboxy-14-(carboxymethyl)-8-(3-guanidinopropyl)-18-hydroxy-3,6,9,12,15-pentaoxo-2-oxa-7,10,13,16-tetraazaoctadecyloxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-iumtrifluoroacetate (84)

A vial was charged with a magnetic stirring bar,7-((8S,14S,17S)-14-(2-tert-butoxy-2-oxoethyl)-17-(tert-butoxymethyl)-20,20-dimethyl-3,6,9,12,15,18-hexaoxo-8-(3-(3-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfonyl)guanidino)propyl)-2,19-dioxa-7,10,13,16-tetraazahenicosyloxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-iumchloride (83) (0.500 g, 0.364 mmol) and a solution of water (20 μL, 1.09mmol) and triisopropyl silane (224 μL, 1.09 mmol) in trifluoroaceticacid (5.19 mL, 70 mmol). The reaction solution was magnetically stirredat room temperature. After 3 hours, the reaction solution was chargedwith ethyl ether (20 mL). The resulting precipitate was filtered, andpurified by high-pressure liquid chromatography to give7-((8S,14S,17S)-17-carboxy-14-(carboxymethyl)-8-(3-guanidinopropyl)-18-hydroxy-3,6,9,12,15-pentaoxo-2-oxa-7,10,13,16-tetraazaoctadecyloxy)-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholinothieno[3,2-b]pyran-4-ium trifluoroacetate(84) as a white solid.

tert-Butoxycarbonyl (Boc) Deprotection Procedure Z Synthesis of5-morpholino-3-(1H-pyrrol-2-yl)-7H-thieno[3,2-b]pyran-7-one (97)

A 20 mL vial was charged with a magnetic stirring bar, tert-butyl2-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)-1H-pyrrole-1-carboxylate(94) (50 mg, 124 μmol), and hydrochloric acid (4.0 M in dioxane, 10 mL).The reaction mixture was magnetically stirred overnight at roomtemperature, concentrated in vacuo, and purified via reverse phasehigh-pressure liquid chromatography to give 5-morpholino-3-(IH-pyrrol-2-yl)-7H-thieno[3,2-b]pyran-7-one (97).

I. Biological Examples Example A. Effect of Test Compounds on DAKTStatus in Prostate Cancer Cells (PC3)

PC3 cells were obtained from the American Type Culture Collection (ATCC,Manassas, Va., Cat.# CRL-1435). Two million cells from the prostatecancer line PC3 were placed into 6 cm culture dishes and allowed to growin complete RPMI 1640 media (Invitrogen, Carlsbad, Calif.,Cat.#22400-105) with 10% fetal bovine serum (Invitrogen, Carlsbad,Calif., Cat.#10438-026). After this time period the cells were serumstarved for 5 hours followed by application of the test compound. Testcompound was added as a DMSO (dimethyl sulfoxide) solution such that thefinal DMSO concentration in the cell media was less than or equal to0.2% by volume After 30 minutes of exposure the growth factor stimulant,human IGF-1 (PeproTech, Inc., Rocky Hill, N.J., Cat.#100-11), was addedto a final concentration of 0.1 μg/ml in each well. After 30 minutes ofIGF-1 exposure, cells were removed from the media and cell lysates wereprepared using RIPA Lysis buffer (Upstate, Lake Placid, N.Y.,Cat.#20-188), keeping on ice. The pAKT serine 473 level was measured induplicate samples of the cell lysates using commercially availableassays such as the Pathscan® Sandwich ELISA kit for Ser473 pAKT (CellSignaling, Danvers, Mass., Cat.#7160). A SpectraMax Plusspectrophotometric plate reader (Molecular Devices, Sunnydale, Calif.)was used to measure the optical density signal for pAKT at 450 nm (OD450nm). The pAKT OD450 nm readings were normalized by total protein amountin the cell lysates determined by standard methods.

Concentrations of test compounds required to inhibit IGF stimulated pAKTlevels to 50% of maximum levels in PC3 cells (termed DM50 for decreasedmaximum 50%) were calculated by inputting the dose responses in thesoftware package GraphPad Prism4 (GraphPad Software, Inc., San Diego,Calif.).

By these methods a number of compounds of the invention were evaluatedand the concentration needed to reduce the detectable pAKT to 50% of themaximal uninhibited signal was calculated. These values (in μMolarunits) are listed in Table 8 under the column heading PC3 pAKT IC50(μMolar).

Example B. Effect of Test Compounds on Prostate Cancer Cell (PC3)Proliferation

PC3 cells were obtained from the American Type Culture Collection (ATCC,Manassas, Va., Cat.# CRL-1435) Two thousand cells from the prostatecancer line PC3 were placed in 50 μL of complete RPMI 1640 media(Invitrogen, Carlsbad, Calif., Cat.#22400-105) with 10% fetal bovineserum (Invitrogen, Carlsbad, Calif., Cat.#10438-026) into each well of96 well cell culture plates in a hexaplicate sampling pattern. A 50 μLaliquot of test compound (prepared from stock test compounds in DMSOdiluted into media) was added to the appropriate well such that thefinal concentrations of test compound were 200, 40, 8, 1.6, 0.32, 0.064,0.0128, 0.00256, and 0 μM and the final DMSO concentration was less thanor equal to 0.2%.

The plates were incubated for 72 hours 37° C. in an atmosphere of 5%CO₂. At the end of this time a 10 μL aliquot of WST solution (RocheApplied Science, Mannheim, Germany) was added into each of the wells.Cells were exposed to the WST solution for 4 hours. A SpectraMax Plusspectrophotometric plate reader (Molecular Devices, Sunnydale, Calif.)was then used to measure the optical density at 450 nm (OD450 nm).Sigmoidal curves were drawn for the dose responses and IC50 values werecalculated using the software package GraphPad Prism4 (GraphPadSoftware, Inc., San Diego, Calif.).

By these methods a number of compounds of the invention were evaluatedand the concentration needed to reduce the prostate cancer cell (PC3)proliferation by 50% was calculated. These values (in μMolar units) arelisted in Table 8 under the column heading IC50 PC3 Proliferation(μMolar).

Example C. Comparison of IC50 Values for LY294002 and Cpd 6

IC50 values for inhibition of PI-3 kinase activity and PC3 cellproliferation were determined for LY294002 and Cpd 6. LY294002 isavailable commercially (CellSignaling Technology, Cat. No. 9901) and iswell described in the literature as a pan-PI-3 kinase inhibitor. IC50values for LY294002 were obtained and are listed in Table 1 below.Likewise, IC50 values for Cpd 6 were also obtained. The fold improvementin the IC50 values (i.e., increased potency) of Cpd 6 in comparison withLY294002 is calculated and shown in Table 1. For the beta and deltaisoforms of PI-3 kinase the increased potency was approximately 2-foldand 4-fold respectively. Additionally, mTOR is a clinically validatedtarget in cancer treatments and Cpd 6 demonstrated almost 2-foldimprovement in inhibiting mTOR versus LY294002. Evaluation of theability of Cpd 6 to block the PI3K pathway and decrease the pAKTactivation in PC3 cells by 50% versus LY294002 demonstrated over a2-fold improvement in activity.

Lastly, the IC50 for inhibition of prostate cancer cell proliferation(PC3 cell line run as described in example A) was determined and islisted in the last row of Table 1. The data clearly shows an almost2-fold improvement in the cellular activity of Cpd 6 versus LY294002.

TABLE 1 Comparison of IC50 values (nM) for LY294002 versus Cpd 6 FoldCategory Enzyme LY294002 Cpd 6 Improvement* PI3K Class 1A p110-alpha 356297 1.2 PI3K Class 1A p110-beta 736 378 1.9 PI3K Class 1A p110-delta3224 784 4.1 PI3K Class 1B p110-gamma 1775 1570 1.1 PI3K Super mTOR 1060610 1.7 Family Enzyme Pathway pAKT 1.3 0.55 2.4 Inhibition Cell PC3 IC507,500-12,100 4,100 1.8-3.0 Proliferation Proliferation *the value forLY294002 is divided by the value obtained for Cpd 6

Several other compounds of the invention had IC50 values against thepanel of enzymes in Table 1 in the range of 1-1 μM, while others hadIC50 values of less than 1 μM (data not shown).

Example D. Therapeutic Effect of Cpd 9 in a Renal Cell CarcinomaXenograft Mouse Model

To determine if Cpd 9 has anti-tumor effects, an in vivo study wasconducted in a 786-0 renal cell carcinoma xenograft model, 786-0 cells(2×10⁶) were inoculated subcutaneously into nude mice in the flank areaand tumor growth was monitored with calipers via external measurements.Tumor volume was calculated using the formula volume=(length×width²)/2.When the average tumor volume reached 400 mm³, mice were randomlydivided into 2 groups and treated with either DMSO (control) or Cpd 9(25 mg/kg, ip) three times weekly (M, W, F) for two weeks. This renalcell carcinoma (RCC) xenograft experiment with nude mice showed thatadministration of Cpd 9 (25 mg/kg, ip, 3×/wk) after about two weeksresulted in 93% tumor growth reduction (p<0.05) compared to the controlgroup. No significant body weight change or other abnormalities wereobserved.

Example E. Therapeutic Effect of Cpd 28 in a Renal Cell CarcinomaXenograft Mouse

To determine if Cpd 28 has anti-tumor effects, an in vivo study wasconducted in a 786-0 renal cell carcinoma xenograft model, 786-0 cells(2×10⁶) were inoculated subcutaneously into nude mice in the flank areaand tumor growth was monitored with calipers via external measurements.Tumor volume was calculated using the formula volume=(length×width²)/2.Treatment was started when tumors reached an average volume of 300 mm³.Mice were randomized into groups of 5 animals each and were givenintraperitoneal administration of either LY294002 (25 mg/kg) or Cpd 28(25 mg/kg or 50 mg/kg) at a frequency of 3 times weekly for 4 weeks.DMSO was used as control since this was the vehicle the compounds weredissolved in. Tumor size was monitored twice weekly and body weight wasmonitored once weekly. Means and standard deviations were calculated andthe Student t test method was used for statistical comparisons. In thesame xenograft model, administration of LY294002 and Cpd 28 at the samedosage (25 mg/kg, ip, 3×/wk) showed that Cpd 28 has a strongerinhibitory tumor growth effect than LY294002 (compared to control) (70%vs. 54% inhibition, respectively) while administration of Cpd 28 attwice the dosage and same administration routine (50 mg/kg, ip, 3×/wk)shows a more potent tumor growth inhibition (compared to control) (81%)also with no significant body weight change or other abnormalitiesnoted.

Example F. Apoptotic Effects of Cpd 9 and Cpd 28 in Renal Cell Carcinoma(786-O)

In order to evaluate the mechanism of action of these compounds, 786-0cell cultures were treated with Cpd 9 or Cpd 28 at differentconcentrations overnight and then analyzed for apoptotic induction. Thecells were stained using standard methods with Annexin V and/orPropedium iodine (PI) at room temperature for 15 minutes. Apoptoticcells at early (Annexin V positive) or late stage (both Annexin V and PIpositive) were detected by Flow Cytometry. The results are shown inTable 2 below and demonstrate a dose dependant increase in apoptosisafter exposure to either Cpd 9 or Cpd 28 under these conditions:

TABLE 2 Dose dependent increase in apoptosis of cancer cells. CpdActinomycin 28 Apoptotic Untreated D Cpd 9 Cpd 9 Cpd 28 (10 cellscontrol (200 ng/mL) (5 μM) (10 μM) (5 μM) μM) Early 3.97 16.93 7.76 8.3110.4 16.45 stage apoptotic Late stage 7.35 12.63 10.5 11.62 11.56 15.43apoptotic Total 11.32 29.56 18.26 19.93 21.96 31.88 Apoptotic Cells

Example G. Cell Proliferation Effects of Cod 28 Across a Broad Spectrumof Cancer Cell Lines

All cell lines were obtained from ATCC and cultured and plated accordingto ATCC recommendations. Cells were cultured in complete culture mediacontaining 10% heat-inactivated fetal bovine serum (FBS, Mediatech,Inc.). For adherent cell lines, once cells reached 70% confluency, theywere trypsinized and resuspended in phenol red free RPMI media(Mediatech, Inc.) plus 10% heat-inactivated FBS (assay buffer) and 100μL aliquots were plated into each well of 96-well microtiter plates at afinal density of 1,000-2,000 cells/well. Cells were incubated overnightat 37° C. in 5% CO₂, and treated with test compounds on the next day.For suspension cell lines, cells were resuspended in assay buffer,plated into 96-well microtiter plates at a density of 10⁴-10⁵cells/well/100 μL, and treatment was started on the same day. Cells weretreated with vehicle (DMSO) and test compounds for 72 hours atconcentration of 100, 70, 40, 20, 10, 5, 2.5, 1.25, 0.5 and 0.2 μM infour replicates at 37° C. in 5% CO₂. Following incubation, viable cellswere quantified by the MTS assay (MTS is:3-[4,5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(Promega (Madison, Wis., USA). At the end of a 72-hour treatment, 100 μLof assay buffer was removed and cells were incubated with 20 μL ofmixture of MTS and phenazine methosulfate (TCI America) at 37° C. in 5%CO₂ for 3 hours. Absorbance values (OD) were measured using a DynexOpsys MR plate reader at a single wavelength of 490 nm. Data wereanalyzed by computer-assisted nonlinear regression to fit the data usingGraphPad Prism (GraphPad software, San Diego, Calif.), IC50 is theconcentration of drug that inhibits cell growth by 50% of the vehicle.Results were expressed as means and are listed in table 3 below.

With the exception of Panc-1 all IC50 values were single digitmicromolar or less. This level of potency was true even for cell linescontaining KRAS mutations (HCT116-colon, A549 lung, BXPC3 pancreatic andRPM18226 myeloma cell lines). Colorectal cancer patients with KRASmutations have been identified as less likely to respond to EGFRinhibitors as reported at ASCO 2008 annual meeting thus representing anunmet medical need. Likewise, Cpd 28 has equal potency on breast cancercell lines MCF-7 and MDA-MB-468 even though the latter cell linerepresents “triple negative” breast cancer. This is a subset of breastcancer that are found to be negative for Her-2, progesterone, andestrogen receptors. This set of breast cancer patients currently havefew good treatment options and represents an unmet medical need.

TABLE 3 IC50 values for Cpd 28 against various cancer cell types. CellLine (Cancer Type) IC50 values of Cpd 28 (μM) A549 (Lung) 2.54 H1299(Lung) 2.43 BXPC3 (Pancreas) 3.35 Panc-1 (Pancreas) 18.42 PC3 (Prostate)1.22 DU145 (Prostate) 2.07 HT29 (Colon) 2.40 Hela (Colon) 2.10 HCT116(Colon) 2.20 MDA-MB468 (Breast) 2.71 MCF7 (Breast) 2.27 U87MG (Brain)1.92 OVCAR3 (Ovary) 1.24 786-O (Renal) 2.82 SKMEL2 (Melanoma) 5.20RPM18226 (Myeloma) 0.73 HL60 (AML) 3.06 K562 (CML) 3.20 HUVEC(Endothelial-noncancer) 1.23

Example H. Blockage of Signal Transduction by Cpd 28 and Cpd 25 inCancer Cell Lines

Angiogenesis is a well-described mechanism of cancer progression andseveral agents are clinically useful due to their ability to inhibitthis process in cancer patients. VEGF is one of the most studiedangiogenic factors. VEGF is secreted by cancer cells in response tohypoxia leading to nearby endothelial cell surface receptor bindingwhich stimulates the endothelial response resulting in new bloodvessels. PI-3 kinase (PI3K) has been shown to be integral to theangiogenic process where inhibition of PI3K blocks hypoxia-induced VEGFproduction in cancer cells as well as the VEGF-mediated stimulation ofendothelial cells. The latter could be due to the ability of PI3Kinhibitors to prevent cell-surface receptor mediated activation of AKTto pAKT and thus block downstream signaling elements. Bv8 is a proteinthat has recently been determined to play a role in angiogenesis andanti-Bv8 antibodies have been shown to inhibit tumor growth in mousestudies (Nature 2007, 450, 825-831). Additionally, recent work (CancerRes. 2008, 68, 5501-5504 and references therein) has indicated that Bv8may play a role in resistance of tumors to treatment with antivascularendothelial growth factor treatment such as antibodies to VEGF. Becauseof this evolving interest in Bv8 we sought to understand the ability ofthe novel PI3K inhibitors of the invention to block Bv8 signaling downthe AKT pathway in comparison to the angiogenic ligand VEGF- andIGF-pathway activation.

To evaluate the effect of Cpd 28 and Cpd 25 on AKT Ser473phosphorylation, two million cells from the prostate cancer line PC3 andrenal carcinoma cell line (786-0) were placed into 6 cm culture dishesand allowed to grow in complete media followed by 5 hours serumstarvation followed by the application of test compound to a finalconcentration of 0, 0.1, 0.5, and 2 μM for 30 minutes. This was followedby the addition of IGF, VEGF and Bv8 to 0.1 μg/mL, 0.1 μg/mL and 2.5μg/mL respectively for another 30 minutes before harvesting the cellsand making cell lysates with RIPA buffer keeping on ice, pAKT levelswere measured in duplicate samples with Cell Signaling's Ser473 pAKTELISA kit. Optical density plate readings of pAKT levels were made at450 nm (OD450 nm) and were normalized by total protein amount of thecell lysates. The dose of compound at which pAKT levels are reduced to50% of the non-treatment level was calculated using GraphPad Prism4software and is listed in Tables 4 and 5 below.

TABLE 4 Concentrations (μM) of Cpd 28 and Cpd 25 Required to InhibitpAKT to Half-maximal Levels Stimulated by Bv8, VEGF and IGF in PC3(prostate cancer) Cell Line Compound Stimulant Cpd 28 Cpd 25 PBS 0.2290.564 Bv8 0.1164 0.9406 VEGF 0.1523 0.3317 IGF 0.888 2.117

TABLE 5 Concentrations (μM) of Cpd 28 and Cpd 25 Required to InhibitpAKT to Half-maximal Levels Stimulated by Bv8, VEGF and IGF in the renalcell carcinoma (786-O) Cell Line Compound Stimulant Cpd 28 Cpd 25 PBS0.5407 0.9176 Bv8 0.3799 0.6994 VEGF 0.3434 0.4976 IGF 2.605 4.389

The above results indicate that in vitro inhibition of PI3K by Cpd 28 orCpd 25 can impede the angiogenic stimulants VEGF and Bv8 in theirability to activate AKT in a dose dependant manner in cancer cells.

Example I. Effect of Test Compounds on pAKT Status in Various CancerCell Lines

Several other cancer cell lines were obtained from ATCC and grownaccording to the supplier's recommendations and treated with testcompounds. DM50 values listed in Table 6 below were obtained by themethods described in Example A:

TABLE 6 Effect of test compounds on DM50 of cancer cells. Type of DM50Cancer Cell Line Cpd 6 Cpd 9 Cpd 28 Non-small H1299 3.192 3.070 0.886cell lung cancer Breast cancer BT-474 1.245 0.906 0.307 Renal cell 786-O4.166 2.662 1.367 carcinoma

Example J. Effect of Test Compounds on Cellular Proliferation in VariousCancer Cell Lines

Several other cancer cell lines were obtained from ATCC and grownaccording to their recommendations and treated with test compounds toobtain the concentration of test compound that inhibited the cellularproliferation by 50% of no-treatment controls. These IC50 values (μM)are listed in Table 7 below obtained by the methods described in ExampleB:

TABLE 7 Inhibition of cancer cell proliferation. Type of IC50 CancerCell Line Cpd 6 Cpd 9 Cpd 28 Non-small H1299 5.024 3.040 0.951 cell lungcancer Breast cancer BT-474 11.62 5.911 3.775 Renal cell 786-O 9.536.238 3.321 carcinoma

Example K. Effect of Test Compounds on Oxidative Stress of ProstateCancer Cells

LNCaP cells were obtained from the American Type Culture Collection(ATCC, Manassas, Va., Cat.# CRL-1740). 3.5 million cells from theprostate cancer line LNCaP were placed into each of three 10 cm culturedishes and allowed to grow overnight in complete RPMI 1640 media(Invitrogen, Carlsbad, Calif., Cat.#22400-105) with 10% fetal bovineserum (Invitrogen, Carlsbad, Calif., Cat#10438-026). Test compound wasadded into the dishes to the concentration of 0 and 20 μM and allowed toincubate at 37° C. in a 5% CO₂ atmosphere for 2 and 24 hours. Followingthis the pH of the cell culture media was adjusted down to 3.5 with 2MHCl. After sitting at 4° C. for 15 minutes, the cell culture media fromeach culture dishes was applied to each of three C18 reverse phasecolumns (Biotage ISOLUTE, Part #220-0020-B, Lot #5223506B2A,www.biotage.com) which were prewashed with 10 mL/column of ethanolfollowed by 10 mL/column of deionized water. The loaded columns werethen washed with 10 mL/column of water, followed by 10 mL/column of 15%ethanol, and finally 10 mL/column hexane. The desired samples were theneluted from the columns by addition of 10 mL/column ethyl acetate. Theethyl acetate was then evaporated to dryness using a rotatingevaporator. Each sample was then dissolved in 500 μL of Assay Bufferprovided in BioMol's Isoprostane Oxidative Stress Assay Kit A (Cat. #AK150-0001, Lot #3-08010825). Using the kit reagents according to themanufacturer's instructions the oxidative product 8-iso-ProstaglandinF2α was quantitated by ELISA. Also, using reagents from the kit astandard curve of oxidation product (8-iso-Prostaglandin F2α) versusoptical density reading at 450 nm. From this curve optical densityreadings from the test samples were converted to μg/mL of the oxidationmarker 8-iso-Prostaglandin F2α. This kit from BioMol is designed todetermine the amount of the 8-iso-Prostaglandin F2α produced as a markerfor the amount of oxidative stress experienced by the cells.

Using this method Cpd 28 gave OD450 nm readings of 1.01 units at 2 hoursand 0.96 units at 24 hours exposure (control=136 units) which translatesto 18.0 and 21.0 μg/mL of 8-iso-Prostaglandin F2α generated respectively(versus control samples of 1.5 μg/mL). Relative to untreated controlsCpd 28 exposure gave an increase of oxidation marker 8-iso-ProstaglandinF2α of 12-fold at 2 hours and 14-fold at 24 hours in prostate cancercells (LNCaP). These results indicate that Cpd 28 quickly induces alarge increase in oxidative stress in prostate cancer cells.

Example L. Effect of Test Compounds in Inhibiting Cancer Stem Cells

A mammosphere cancer stem cell inhibition assay was set up usingliterature methods (T. M. Phillips et al. J. Natl. Cancer I. 2006, 98,1777-1785, “Mammalian target of rapamycin contributes to the acquiredapoptotic resistance of human mesothelioma multicellular spheroids”)using a subset of MCF7 breast cancer cells that posses the ability toevade anchorage independent apoptosis. Commercially available MCF7breast cancer cells were cultured in MEM supplemented with 0.01 mg/mL ofbovine insulin and 10% FBS at 37° C. in humidified atmosphere (5% CO₂).Floating cells in the 2-day cultures were collected, washed, andresuspended in DMEM-F12 (50:50) supplemented with 0.4% BSA, 5 μg/mLbovine insulin, 20 ng/mL bFGF, and 10 ng/mL EGF. Cells were then passedthrough a 40 μM sieve to remove any cell clusters and adjusted to adensity of 10,000 cells/mL. Approximately 500 cells were plated inquadruplicate in a sterile poly-HEMA-treated 96-well plate. Cells werethen treated with either control or test solutions consisting of thekinase inhibitor Lapatinib (1 μM), Cpd 28 (1 or 10 μM), Cpd 55 (1 or 10μM), The numbers of mammospheres (clusters of cells numbering more than4 or 5 per cluster) were then counted after 5-7 days.

In one experiment for the control sample (no test agent added) a mean ofthe quadruplicate measurements yielded 38±4 mammospheres whereas thekinase inhibitor Lapatinib, approved for treating breast cancer, at 1 μMgave 26±2 mammospheres. Cpd 28 at 1 μM or 10 μM yielded 15±2 and 5±1mammospheres respectively. Cpd 55 at 1 μM or 10 μM yielded 16±2 and 5±1mammospheres respectively. Thus, Cpd 28 and Cpd 55 at 1 μM gaveapproximately 60% inhibition of mammosphere production versus controlwhereas Lapatinib at this same concentration only gave 33% inhibition ofmammosphere production. At the higher concentration of 10 μM Cpd 28 andCpd 55 gave approximately 88% and 86% inhibition of mammosphereproduction versus control, respectively.

In a separate experiment for the control sample (no test agent added) amean of the quadruplicate measurements yielded 32±6 mammospheres whereasthe kinase inhibitor Lapatinib, approved for treating breast cancer, at1 uM gave 17±4 mammospheres. Cpd 25 at 1 μM or 10 μM yielded 12±3 and7±2 mammospheres respectively. Cpd 43 at 1 μM or 10 μM yielded 18±6 and16±6 mammospheres respectively. Thus, Cpd 25 and Cpd 43 at 1 μM gaveapproximately 69 and 53% inhibition (respectively) of mammosphereproduction versus control whereas Lapatinib at this same concentrationgave 55% inhibition of mammosphere production. At the higherconcentration of 10 μM Cpd 25 and Cpd 43 gave approximately 82% and 58%inhibition of mammosphere production versus control, respectively.

Representative compounds of the invention along with NRM spectral dataare depicted in Tables 8 and 9 respectively as merely illustrative andnot limiting of the scope of the invention in any way.

TABLE 8 Illustrative compounds of formula I. IC50 Com- PC3 PC3 Syn-pound pAKT Prolif- Mass thetic num- IC50 eration Ex- Mass Meth- berStructure (μMolar) (μMolar) pected Found od  1

0.864  20.18   327 328 (M + H) B  2

3.031  18.5    314 356 (M + H + CH3CN) B  3

4.872  10.88   314 315 (M + H) B  4

0.93    5.9    303 304 (M + H) A  5

7.3  ND  338 339 (M + H) A  6

0.55    4.11   313 314 (M + H) A  7

0.54    1.74   357 358 (M + H) A  8

0.98    1.82   343 344 (M + H) A  9

0.78    2.01   356 357 (M + H) A  10

ND ND  237 238 (M + H) B  11

6.317   6.293  328 329 (M + H) B  12

1.574   8.552  352 353 (M + H) B  13

5.295  15.31   391 392 (M + H) A  14

1.021   4.362  319 320 (M + H) A  15

0.948   4.475  327 328 (M + H) A  16

1.229   6.028  327 328 (M + H) A  17

1.41   28.96   347 348 (M + H) A  18

0.913  37.62   381 382 (M + H) A  19

0.901   8.132  352 353 (M + H) B  20

ND 774.5    381 382 (M + H) B  21

1.038  34.81   393 392 (M − H) E  22

0.609   1.054  384 385 (M + H) A  23

1.274   7.347  343 344 (M + H) A  24

4.606  28.17   291 292 (M + H) A  25

ND   1.233-   3.141  385 386 (M + H) B  26

1.558   5.597  319 320 (M + H) A  27

3.235   9.26  303 304 (M + H) A  28

0.161- 0.44    1.298-   1.914  371 372 (M + H) A  29

1.067   4.759  343 344 (M + H) A  30

ND   5.799  433 434 (M + H) F  31

ND   3.689  337 338 (M + H) C  32

ND   9.008  348 349 (M + H) A  33

ND ND  475 474 (M − H) F  34

ND   2      432 431 (M − H) F  35

ND   2.699-   5.59   352 353 (M + H) A  36

ND   2.694  442 443 (M + H) A  37

ND   2.496  380 381 (M + H) C  38

0.363   8.421  367 368 (M + H) C  39

ND   3.31   355 356 (M + H) C  40

ND   1.803  397 398 (M + H) C  41

ND   2.067  342 343 (M + H) A  42

ND   7.844  369 370 (M + H) A  43

ND   9.351  413 414 (M + H) A  44

ND   4.394  371 372 (M + H) B  45

ND   4.673  371 372 (M + H) B  46

ND  62.15   433 434 (M + H) F  47

ND ND  350 351 (M + H) D  48

ND ND  433 434 (M + H) F  49

ND   3.181  475 474 (M − H) F  50

ND   4.764  446 447 (M + H) G  51

ND  23.77   356 357 (M + H) D  52

ND 157.1    336 337 (M + H) D  53

ND 112.8    370 371 (M + H) D  54

ND ND  432 433 (M + H) G  55

ND   1.053-   1.504  433 434 (M + H) F  56

ND ND  328 329 (M + H) A  57

ND ND  353 354 (M + H) C  58

ND  16.74   433 434 (M + H) G  59

ND  17.49   433 434 (M + H) G  60

ND ND  475 476 (M + H) G  61

ND   4.999  433 434 (M + H) G  62

ND ND  387 388 (M + H) I  63

ND   7.841  446 447 (M + H) G  64

ND  17.89   433 434 (M + H) G  65

ND  12.66   475 476 (M + H) G  66

ND   6.905  433 434 (M + H) G  67

ND  95.84   306 307 (M + H) H  68

ND  34.81   323 324 (M + H) I  69

ND ND  282 283 (M + H) E  70

ND ND  281 208 (M − H) J  71

ND ND  320 321 (M + H) K  72

ND ND  398 399 (M + H) L  73

ND ND  357 358 (M + H) M  74

ND ND  279 280 (M + H) N  75

ND ND  413 414 (M + H) O  76

ND ND  391 392 (M + H) P  77

ND ND  470 471 (M + H) R  78

ND ND  326 326 (M+) S  79

ND ND  366 366 (M+) T  80

ND ND  336 336 (M+) 377 (M + CH3CN) U  81

ND ND  558 558 (M+) V  82

ND ND ND ND W  83

ND ND 1337 669 ([M/Z] + 1) (Z = 2) X  84

ND ND  917 459 ([M/Z] + 1) (Z = 2) Y  85

ND   3.452  347 348 (M + H) A  86

ND ND  358 359 (M + H) B  87

ND ND  370 371 (M + H) F  88

ND  21.97   404 405 (M + H) D  89

ND ND  372 373 (M + H) D  90

ND  19.93   393 394 (M + H) D  91

ND ND  294 295 (M + H) D  92

ND   6.281  381 382 (M + H) A  93

ND   6.14-    9.431  363 364 (M + H) A  94

ND ND  402 403 (M + H) A  95

ND  81.87   358 359 (M + H) A  96

ND  24.84   352 353 (M + H) C  97

ND ND  302 303 (M + H) Z  98

ND   6.124  329 330 (M + H) A  99

ND  16.6    329 330 (M + H) A 100

ND 128.9    357 358 (M + H) 356 (M − H) B 101

ND  21.76   392 392 (M+) E 102

ND ND  412 413 (M + H) A 103

ND ND  316 316 (M+) A 104

ND ND  372 373 (M + H) A 105

ND ND  344 345 (M + H) A 106

ND ND  401 402 (M + H) A 107

ND ND  359 360 (M + H) A 108

ND ND  348 349 (M + H) A 109

ND ND  357 356 (M − H) B 110

ND ND  393 394 (M + H) A 111

ND ND  377 378 (M + H) A 112

ND ND  377 378 (M + H) A 113

ND ND  343 344 (M + H) A 114

ND ND  365 366 (M + H) A 115

ND ND  361 362 (M + H) A 116

ND ND  353 354 (M + H) B 117

ND ND  391 392 (M + H) B ND-not determined

TABLE 9 NMR Data of representative compounds Cpd No. NMR Analytical Data2 ¹H NMR (DMSO-d₆): 8.70 (dd, J = 4.4, 1.6 Hz, 2H), 8.47 (s, 1H), 7.74(dd, J = 4.4, 1.6 Hz, 2H), 5.57 (s, 1H), 3.73 (t, J = 5.0 Hz, 4H), 3.45(t, J = 4.9 Hz, 4H). 3 ¹H NMR (DMSO-d₆): 8.94 (dd, J = 2.3, 0.9 Hz, 1H),8.61 (dd, J = 4.9, 1.6 Hz, 1H), 8.33 (s, 1H), 8.14 (ddd, J = 7.8, 2.3,1.6 Hz, 1H), 7.55 (ddd, J = 7.8, 4.9, 0.9 Hz, 1H), 5.56 (s, 1H), 3.71(t, J = 4.9 Hz, 4H), 3.42 (t, J = 5.0 Hz, 4H). 4 ¹H NMR (DMSO-d₆): 8.13(s, 1H), 7.83 (dd, J = 1.9, 0.7 Hz, 1H), 6.84 (dd, J = 3.4, 0.6 Hz, 1H),6.66 (dd, J = 3.4, 1.7 Hz, 1H), 5.55 (s, 1H), 3.75 (t, J = 5.0 Hz, 4H),3.50 (t, J = 4.9 Hz, 4H). 6 ¹H NMR (DMSO-d₆): 8.18 (s, 1H), 7.72 (dd, J= 8.5, 1.3 Hz, 2H), 7.51 (dd, J = 8.1, 7.0 Hz, 2H), 7.42 (d, J = 7.4 Hz,1H), 5.54 (s, 1H), 3.71 (t, J = 5.0 Hz, 4H), 3.43 (t, J = 4.9 Hz, 4H).h7 ¹H NMR (DMSO-d₆): 8.08 (s, 1H), 7.28 (d, J = 1.6 Hz, 1H), 7.22 (dd, J= 8.0, 1.7 Hz, 1H), 7.04 (d, J = 8.1 Hz, 1H), 6.08 (s, 2H), 5.53 (s,1H), 3.71 (t, J = 5.0 Hz, 4H), 3.42 (t, J = 5.0 Hz, 4H). 8 ¹H NMR(DMSO-d₆): 8.06 (s, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 9.0 Hz,2H), 5.53 (s, 1H), 3.80 (s, 3H), 3.72 (t, J = 4.9 Hz, 4H), 3.43 (t, J =5.0 Hz, 4H). 9 ¹H NMR (DMSO-d₆): 7.94 (s, 1H), 7.57-7.53 (m, 2H), 6.81(d, J = 9.0 Hz, 2H), 5.52 (s, 1H), 3.72 (t, J = 5.0 Hz, 4H), 3.44 (t, J= 4.9 Hz, 4H), 2.50 (s, 6H). 11 ¹H NMR (DMSO-d₆): 8.01 (s, 1H), 7.12 (t,J = 7.9 Hz, 1H), 6.87 (t, J = 1.9 Hz, 1H), 6.81 (dd, J = 8.5, 0.8 Hz,1H), 6.59 (ddd, J = 8.0, 2.2, 1.0 Hz, 1H), 5.52 (s, 1H), 5.25 (s, 2H),3.72 (t, J = 4.9 Hz, 4H), 3.43 (t, J = 5.0 Hz, 4H). 13 ¹H NMR (DMSO-d₆):8.40 (s, 1H), 8.08-7.92 (m, 4H), 5.57 (s, 1H), 3.73-3.71 (m, 4H),3.46-3.43 (m, 4H), 3.28 (s, 3H). 14 ¹H NMR (DMSO-d₆): 8.23 (s, 1H), 7.62(ddd, J = 8.8, 4.4, 2.3 Hz, 4H), 7.21 (dd, J = 5.1, 3.7 Hz, 1H), 5.55(s, 1H), 3.75 (t, J = 5.0 Hz, 4H), 3.52 (t, J = 5.0 Hz, 4H). 15 ¹H NMR(DMSO-d₆): 8.12 (s, 1H), 7.61 (d, J = 8.1 Hz, 2H), 7.32 (dd, J = 8.5,0.6 Hz, 2H), 5.53 (s, 1H), 3.71 (t, J = 5.0 Hz, 4H), 3.42 (t, J = 5.0Hz, 4H), 2.35 (s, 3H). 16 ¹H NMR (DMSO-d₆): 8.16 (s, 1H), 7.55-7.49 (m,2H), 7.39 (t, J = 7.5 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H), 5.54 (s, 1H),3.72 (t, J = 5.0 Hz, 4H), 3.44 (t, J = 4.9 Hz, 4H), 2.37 (s, 3H). 19 ¹HNMR (DMSO-d₆): 11.33 (s, 1H), 8.11 (s, 1H), 7.75 (t, J = 0.7 Hz, 1H),7.65 (d, J = 8.1 Hz, 1H), 7.44 (t, J = 2.8 Hz, 1H), 7.35 (dd, J = 8.2,1.5 Hz, 1H), 6.48-6.46 (m, 1H), 5.55 (s, 1H), 3.73 (t, J = 4.9 Hz, 4H),3.46 (t, J = 4.9 Hz, 4H). 26 ¹H NMR (DMSO-d₆): 8.21 (s, 1H), 7.89 (dd, J= 2.9, 1.3 Hz, 1H), 7.71 (dd, J = 5.0, 2.9 Hz, 1H), 7.57 (dd, J = 5.0,1.3 Hz, 1H), 5.54 (s, 1H), 3.74 (t, J = 5.0 Hz, 4H), 3.47 (t, J = 5.0Hz, 4H). 27 ¹H NMR (DMSO-d₆): 8.12 (s, 2H), 7.83 (t, J = 1.7 Hz, 1H),7.00 (dd, J = 1.9, 0.9 Hz, 1H), 5.53 (s, 1H), 3.74 (t, J = 5.0 Hz, 4H),3.46 (t, J = 5.0 Hz, 4H). 28 ¹H NMR (DMSO-d₆): 8.07 (s, 1H), 7.22-7.19(m, 2H), 6.98 (dd, J = 8.0, 0.8 Hz, 1H), 5.53 (s, 1H), 4.28 (s, 4H),3.71 (t, J = 5.0 Hz, 4H), 3.43 (t, J = 4.9 Hz, 4H). 29 ¹H NMR (DMSO-d₆):8.20 (s, 1H), 7.42 (t, J = 8.2 Hz, 1H), 7.30-7.28 (m, 2H), 6.98 (ddd, J= 4.6, 2.3, 2.3 Hz, 1H), 5.54 (s, 1H), 3.80 (s, 3H), 3.71 (t, J = 5.0Hz, 4H), 3.44 (t, J = 4.9 Hz, 4H). 31 ¹H NMR (DMSO-d₆): 8.31 (s, 1H),7.59-7.57 (m, 2H), 7.48-7.44 (m, 3H), 5.53 (s, 1H), 3.74 (t, J = 4.9 Hz,4H), 3.49 (t, J = 4.9 Hz, 4H). 32 ¹H NMR (DMSO-d₆): 8.79 (dd, J = 2.6,0.7 Hz, 1H), 8.36 (s, 1H), 8.21 (dd, J = 8.3, 2.5 Hz, 1H), 7.69 (dd, J =8.5, 0.6 Hz, 1H), 5.56 (s, 1H), 3.72 (t, J = 4.9 Hz, 4H), 3.42 (t, J =4.9 Hz, 4H). 35 ¹H NMR (DMSO-d₆): 11.34 (s, 1H), 8.10 (s, 1H), 7.76 (s,1H), 7.64 (d, J = 8.1 Hz, 1H), 7.44 (t, J = 2.7 Hz, 1H), 7.34 (dd, J =8.2, 1.5 Hz, 1H), 6.47 (s, 1H), 5.54 (s, 1H), 3.73 (t, J = 4.9 Hz, 4H),3.46 (t, J = 5.0 Hz, 4H). 36 ¹H NMR (DMSO-d₆): 8.16 (s, 1H), 7.68 (d, J= 8.8 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 5.54 (s, 1H), 3.71 (t, J = 4.9Hz, 4H), 3.43 (t, J = 4.9 Hz, 4H), 3.23 (s, 3H), 1.41 (s, 9H). 37 ¹H NMR(DMSO-d₆): 8.15 (s, 1H), 7.36 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 9.0 Hz,2H), 5.52 (s, 1H), 3.74 (t, J = 4.9 Hz, 4H), 3.49 (t, J = 4.9 Hz, 4H),2.96 (s, 6H). 38 ¹H NMR (DMSO-d₆): 8.24 (s, 1H), 7.51 (d, J = 8.8 Hz,2H), 7.01 (d, J = 8.8 Hz, 2H), 5.52 (s, 1H), 3.80 (s, 3H), 3.74 (t, J =4.9 Hz, 4H), 3.48 (t, J = 4.9 Hz, 4H). 39 ¹H NMR (DMSO-d₆): 8.34 (s,1H), 7.54-7.30 (m, 4H), 5.54 (s, 1H), 3.74 (t, J = 4.9 Hz, 4H), 3.49 (t,J = 4.9 Hz, 4H). 40 ¹H NMR (DMSO-d₆): 8.30 (s, 1H), 6.71 (d, J = 2.3 Hz,2H), 6.60 (t, J = 2.3 Hz, 1H), 5.53 (s, 1H), 3.77 (s, 6H), 3.73 (t, J =4.9 Hz, 4H), 3.49 (t, J = 4.9 Hz, 4H). 41 ¹H NMR (CDCl₃): 7.48-7.38 (m,4H), 6.72-6.62 (m, 2H), 5.50 (s, 1H), 3.79 (s, 4H), 3.38 (s, 4H), 2.85(s, 3H). 42 ¹H NMR (CDCl₃): 8.12 (s, 2H), 8.02 (s, 1H), 7.84-7.70 (m,2H), 7.58-7.48 (m, 1H), 5.58 (s, 1H), 4.15-3.88 (m, 10H), 3.48 (s, 4H),3.06-2.90 (m, 6H). 43 ¹H NMR (CDCl₃): 8.59 (s, 2H), 8.32 (s, 1H), 7.57(s, 1H), 5.54 (s, 1H), 4.03 (s, 4H), 3.82 (s, 4H), 3.41 (s, 4H), 2.78(s, 4H), 2.54 (s, 3H). 44 ¹H NMR (CDCl₃): 8.18-8.10 (m, 2H), 7.75-7.71(m, 3H), 5.50 (s, 1H), 3.96 (s, 3H), 3.80 (s, 4H), 3.42 (s, 4H). 45 ¹HNMR (CDCl₃): 8.42 (s, 1H), 8.08-8.04 (m, 1H), 7.82-7.78 (m, 1H), 7.72(s, 1H), 7.62-7.55 (m, 1H), 5.50 (s, 1H), 3.94 (s, 3H), 3.84 (s, 4H),3.50 (s, 4H). 47 ¹H NMR (CDCl₃): 7.70 (s, 1H), 5.48 (s, 1H), 3.95-3.30(m, 16H). 51 ¹H NMR (CDCl₃): 8.24 (s, 1H), 8.18 (s, 1H), 7.64-7.20 (m,5H), 5.46 (s, 1H), 3.82 (s, 4H), 3.46 (s, 4H). 52 ¹H NMR (CDCl₃): 7.62(s, 1H), 5.48 (s, 1H), 3.94-3.22 (m, 18H). 53 ¹H NMR (DMSO-d₆): 8.16 (s,1H), 7.42-7.23 (m, 5H), 6.48 (s, 1H), 5.40 (s, 1H), 4.60 (s, 2H), 3.58(s, 4H), 3.14 (s, 4H). 55 ¹H NMR (CD₃OD): 9.11 (s, 1H), 8.78 (s, 1H),8.40 (s, 1H), 7.99 (s, 1H), 7.89-7.70 (m, 6H), 5.60 (s, 1H), 3.56 (s,4H), 3.31 (s, 4H). 56 ¹H NMR (DMSO-d₆): 7.861 (s, 1H), 7.37 (d, J = 8.6Hz, 2H), 6.64 (d, J = 86 Hz, 2H), 5.50 (s, 1H), 5.35 (s, 2H), 3.72 (t, J= 4.9 Hz, 4H), 3.42 (t, J = 4.9 Hz, 4H). 57 ¹H NMR (DMSO-d₆): 9.79 (s,1H), 8.29 (s, 1H), 7.24 (t, J = 8.0 Hz, 1H), 6.99 (dt, J = 2.3, 1.2 Hz,1H), 6.91 (t, J = 1.9 Hz, 1H), 6.86 (ddd, J = 5.5, 2.7, 1.5 Hz, 1H),5.53 (s, 1H), 3.74 (t, J = 4.9 Hz, 4H), 3.49 (t, J = 4.9 Hz, 4H). 58 ¹HNMR (DMSO-d₆): 10.57 (s, 1H), 8.97 (s, 1H), 8.35 (s, 2H), 8.25-8.13 (m,3H), 7.93 (d, J = 9 Hz, 2H), 7.48-7.39 (m, 1H), 5.57 (s, 1H), 3.74 (s,4H), 3.47 (s, 4H). 59 ¹H NMR (DMSO-d₆): 10.70 (s, 1H), 8.50 (d, J = 6.0Hz, 2H), 8.35 (s, 1H), 8.14 (d, J = 9.0 Hz, 2H), 7.93 (d, J = 9 Hz, 2H),7.82 (d, J = 6.0 Hz, 2H), 5.57 (s, 1H), 3.74 (s, 4H), 3.46 (s, 4H). 60¹H NMR (DMSO-d₆): 10.12 (s, 1H), 8.32 (s, 1H), 8.11 (d, J = 8.9 Hz, 2H),7.87 (d, J = 6.0 Hz, 2H), 7.61 (d, J = 9.0 Hz, 2H), 6.74 (d, J = 6.0 Hz,2H), 5.56 (s, 1H), 3.74 (s, 4H), 3.45 (s, 4H), 2.88 (s, 6H). 61 ¹H NMR(DMSO-d₆): 10.87 (s, 1H), 8.41 (d, J = 6.0 Hz, 1H), 8.35 (s, 1H),8.20-8.16 (m, 2H), 7.88 (d, J = 8.2 Hz, 4H), 7.19 (dd, J = 6.6, 4.8 Hz,1H), 5.57 (s, 1H), 3.74 (s, 4H), 3.46 (s, 4H). 63 ¹H NMR (DMSO-d₆): 9.15(t, J = 5.9 Hz, 1H), 8.35 (s, 1H), 8.29 (s, 1H), 8.04-7.85 (m, 2H), 7.62(t, J = 9.0 Hz, 2H), 7.33 (s, 3H), 7.30-7.17 (m, 1H), 5.55 (s, 1H), 4.51(d, J = 6.0 Hz, 2H), 3.66 (s, 4H), 3.44 (s, 4H). 64 ¹H NMR (DMSO-d₆):10.70 (s, 1H), 8.51 (d, J = 5.6 Hz, 2H), 8.42 (s, 1H), 8.37 (s, 1H),8.02 (t, J = 6.2 Hz, 2H), 7.81 (d, J = 5.9 Hz, 2H), 7.70 (t, J = 6.0 Hz,1H), 5.56 (s, 1H), 3.69 (s, 4H), 3.48 (s, 4H). 65 ¹H NMR (DMSO-d₆):10.08 (s, 1H), 8.40 (s, 1H), 8.34 (s, 1H), 7.96 (dd, J = 16.5, 7.5 Hz,2H), 7.65 (t, J = 7.9 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 6.75 (d, J =8.8 Hz, 2H), 5.56 (s, 1H), 3.68 (s, 4H), 3.48 (s, 4H), 2.88 (s, 6H). 66¹H NMR (DMSO-d₆): 10.92 (s, 1H), 8.51 (s, 1H), 8.47-8.38 (m, 2H), 8.23(d, J = 9.0 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 7.3 Hz, 1H),7.94-7.81 (m, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.19 (t, J = 6.0 Hz, 1H),5.57 (s, 1H), 3.69 (s, 4H), 3.48 (s, 4H). 67 ¹H NMR (CD₃OD): 8.12 (dd, J= 8.1, 1.6 Hz, 1H), 7.78 (dd, J = 7.5, 1.6 Hz, 1H), 7.64-7.40 (m, 6H),6.29 (s, 1H), 3.97 (dd, J = 11.4, 2.6 Hz, 2H), 3.48 (dt, J = 11.6, 2.3Hz, 2H), 2.89 (tt, J = 11.5, 4.0 Hz, 1H), 1.89-1.63 (m, 4H). 68 ¹H NMR(DMSO-d₆): 8.50 (dd, J = 8.0, 1.7 Hz, 1H), 7.69-7.45 (m, 7H), 6.90 (s,1H), 3.67 (t, J = 4.9 Hz, 4H), 3.50 (t, J = 4.9 Hz, 4H). 81 ¹H NMR(CDCl₃): 7.82 (s, 1H), 7.45 (s, 1H), 7.04 (s, 1H), 6.58 (s, 2H), 4.43(s, 2H), 4.32 (s, 6H), 4.08 (s, 2H), 3.92 (s, 4H), 2.73 (t, J = 6.8 Hz,2H), 2.58 (t, J = 6.8 Hz, 2H), 1.41 (s, 9H). 85 ¹H NMR (DMSO-d₆): 8.29(s, 1H), 7.81 (s, 1H), 7.70 (d, J = 7.3 Hz, 1H), 7.57-7.47 (m, 2H), 5.54(s, 1H), 3.73 (s, 4H), 3.44 (s, 4H). 86 ¹H NMR (DMSO-d₆): 8.65 (t, J =2.0 Hz, 1H), 8.48 (s, 1H), 8.26-8.21 (m, 2H), 7.82 (t, J = 8.1 Hz, 1H),5.58 (s, 1H), 3.73 (t, J = 4.9 Hz, 4H), 3.49 (t, J = 4.9 Hz, 4H). 88 ¹HNMR (CDCl₃): 8.29 (s, 1H), 7.35-7.30 (m, 4H), 6.46 (s, 1H), 5.43 (s,1H), 4.60 (d, J = 5.3 Hz, 2H), 3.65 (t, J = 4.5 Hz, 4H), 3.17 (t, J =4.5 Hz, 4H). 90 ¹H NMR (CDCl₃): 8.25 (s, 1H), 7.76 (s, 1H), 5.55 (s,1H), 3.72-3.54 (m, 8H), 2.95-2.73 (m, 12H). 91 ¹H NMR (CDCl₃): 8.10 (s,1H), 6.26 (s, 1H), 5.47 (s, 1H), 3.85 (d, J = 3.0 Hz, 3H), 3.47 (s, 4H),3.03 (d, J = 4.8 Hz, 4H). 92 ¹H NMR (CD₃OD): 8.16 (s, 1H), 8.09 (s, 1H),7.96 (s, 1H), 7.72 (s, 2H), 5.59 (s, 1H), 3.80 (s, 4H), 3.54 (s, 4H). 93¹H NMR (CD₃OD): 7.92 (s, 1H), 7.85 (s, 1H), 7.48 (s, 1H), 7.05 (s, 1H),5.58 (s, 1H), 3.84 (s, 4H), 3.59 (s, 4H). 94 ¹H NMR (CDCl₃): 7.46 (d, J= 11.6 Hz, 2H), 6.31 (d, J = 6.9 Hz, 2H), 5.49 (s, 1H), 3.76 (s, 4H),3.31 (s, 4H), 1.30 (s, 9H). 95 ¹H NMR (CDCl₃): 7.48 (s, 1H), 7.04-6.99(m, 2H), 6.78 (d, J = 7.8 Hz, 1H), 5.51 (s, 1H), 3.95-3.78 (m, 7H), 3.44(s, 4H). 96 ¹H NMR (CDCl₃): 7.55 (s, 1H), 7.27 (s, 2H), 7.22-7.18 (m,1H), 6.95-6.65 (m, 1H), 5.46 (s, 1H), 3.85 (s, 4H), 3.51 (s, 4H). 97 ¹HNMR (CDCl₃): 8.96 (s, 1H), 7.55 (s, 1H), 6.93 (s, 1H), 6.53 (s, 1H),6.33 (s, 1H), 5.48 (s, 1H), 3.85 (s, 4H), 3.49 (s, 4H). 98 ¹H NMR(CDCl₃): 7.52-7.45 (m, 2H), 6.97-6.92 (m, 2H), 5.60 (s, 1H), 5.50 (s,1H), 3.80 (s, 4H), 3.42 (s, 4H). 99 ¹H NMR (CDCl₃): 7.60 (s, 1H),7.40-7.25 (m, 3H), 7.15 (s, 1H), 5.49 (s, 1H), 3.80 (s, 4H), 3.50 (s,1H), 3.43 (s, 4H). 100 ¹H NMR (DMSO-d₆): 8.30 (s, 1H), 8.05 (d, J = 8.2Hz, 2H), 7.81 (d, J = 7.9 Hz, 2H), 5.55 (s, 1H), 3.72 (s, 4H), 3.44 (s,4H). 101 ¹H NMR (DMSO-d₆): 8.24 (s, 1H), 7.82-7.65 (m, 4H), 5.55 (s,1H), 3.72 (s, 4H), 3.43 (s, 4H). 102 ¹H NMR (DMSO-d₆): 8.16 (d, J = 6.0Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 7.9 Hz, 2H), 5.53 (s,1H), 3.73 (s, 4H), 3.61 (s, 4H), 3.57 (s, 2H), 3.42 (s, 4H), 2.40 (s,4H). 104 ¹H NMR (DMSO-d₆): 9.10 (s, 1H), 8.52-8.48 (m, 1H), 8.34-8.28(m, 1H), 8.18-8.10 (m, 1H), 5.55 (s, 1H), 3.95 (s, 3H), 3.75 (s, 4H),3.46 (s, 4H). 105 ¹H NMR (DMSO-d₆): 8.53 (s, 1H), 8.18 (s, 1H), 8.06 (d,J = 6.6 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 5.55 (s, 1H), 3.91 (s, 3H),3.72 (s, 4H), 3.42 (s, 4H). 106 ¹H NMR (CDCl₃): 7.91 (d, J = 6.0 Hz,1H), 7.69 (s, 1H), 7.27-7.18 (m, 2H), 5.50 (s, 1H), 3.96 (s, 3H), 3.93(s, 3H), 3.82 (s, 4H), 3.43 (s, 4H). 107 ¹H NMR (CDCl₃): 7.52 (s, 1H),7.12-7.00 (m, 3H), 5.88 (s, 1H), 5.49 (s, 1H), 3.93 (s, 3H), 3.81 (s,4H), 3.43 (s, 4H).

What is claimed is:
 1. A method for treating cancer in a patient in needthereof comprising the steps of: a. determining inhibition ofproliferation of one or more cancer cell types in vitro comprisingcontacting said cell types with a compound of the formula I, or apharmaceutically acceptable salt thereof,

wherein M is O or S; R1 is selected from H, F, Cl, Br, I, alkenyl,alkynyl, carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro,cyano, amino, carboxylic acid, carboxylic ester, carboxyl amide, reversecarboxyamide, substituted alkyl, substituted alkenyl, substitutedalkynyl, substituted carbocycle, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate; R2 is selected from H, F, Cl, Br, I,alkyl, alkenyl, alkynyl, carbocycle, heteroaryl, formyl, nitro, cyano,carboxylic acid, carboxylic ester, carboxyl amide, reverse carboxyamide,substituted alkenyl, substituted alkynyl, substituted carbocycle,substituted heterocycle, substituted heteroaryl, phosphonic acid,phosphinic acid, phosphoramidate, phosphonic ester, phosphinic ester,ketone, substituted ketone, hydroxamic acid, N-substituted hydroxamicacid, O-substituted hydroxamate, N- and O-substituted hydroxamate,sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfonicacid, sulfonic ester, sulfonamide, N-substituted sulfonamide,N,N-disubstituted sulfonamide, boronic acid, boronic ester, azo,substituted azo, azido, nitroso, imino, substituted imino, oxime,substituted oxime, alkoxy, substituted alkoxy, aryloxy, substitutedaryloxy, thioether, substituted thioether, carbamate, substitutedcarbamate; or

Where X is C, N, P, P(O), SiR^(b); n is 0, 1 or 2; Y is C—R1, O, S,NR^(a), —C(O)(NH₂), —P(Z)mR^(a), SiR^(a)R^(b), BR^(b); Z is O or S; m is0 or 1; R^(a) is hydrogen (H) or independently at each instance anygroup selected from H, F, Cl, Br, I, alkyl, alkenyl, alkynyl,carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino,carboxylic acid, carboxylic ester, carboxyl amide, reverse carboxyamide,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedcarbocycle, substituted aryl, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate; R^(b) is hydrogen (H) or independentlyat each instance any group selected from F, Cl, Br, I, alkyl, alkenyl,alkynyl, carbocycle, aryl, heterocycle, heteroaryl, formyl, cyano,amino, carboxylic acid, carboxylic ester, carboxyl amide, reversecarboxyamide, substituted alkyl, substituted alkenyl, substitutedalkynyl, substituted carbocycle, substituted aryl, substitutedheterocycle, substituted heteroaryl, phosphonic acid, phosphinic acid,phosphoramidate, phosphonic ester, phosphinic ester, ketone, substitutedketone, hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate; R3 is selected from H, F, Cl, Br, I,alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocycle, heteroaryl,formyl, nitro, cyano, amino, carboxylic acid, carboxylic ester, carboxylamide, reverse carboxyamide, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted carbocycle, substituted aryl,substituted heterocycle, substituted heteroaryl, phosphonic acid,phosphinic acid, phosphoramidate, phosphonic ester, phosphinic ester,ketone, substituted ketone, hydroxamic acid, N-substituted hydroxamicacid, O-substituted hydroxamate, N- and O-substituted hydroxamate,sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfonicacid, sulfonic ester, sulfonamide, N-substituted sulfonamide,N,N-disubstituted sulfonamide, boronic acid, boronic ester, azo,substituted azo, azido, nitroso, imino, substituted imino, oxime,substituted oxime, alkoxy, substituted alkoxy, aryloxy, substitutedaryloxy, thioether, substituted thioether, carbamate, substitutedcarbamate; R4 is selected from H, F, Cl, Br, L alkyl, alkenyl, alkynyl,carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino,carboxylic acid, carboxylic ester, carboxyl amide, reverse carboxyamide,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedcarbocycle, substituted aryl, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oximo, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate; and Cyc is an aryl, substituted aryl,heterocycle, substituted heterocycle, carbocycle, and substitutedcarbocycle, and monitoring the growth of said cancer cell types versus anon-treatment control group; and b. administering to said patient aneffective amount of a compound identified in step a as being effectivein inhibiting proliferation of cancer cells in vitro that are of thesame type as said cancer in said patient.
 2. A method of treating cancerin a patient in need thereof comprising administering to said patient aneffective amount of a compound of formula I, or a pharmaceuticallyacceptable salt thereof,

wherein M is O or S; R1 is selected from H, F, Cl, Br, I, alkenyl,alkynyl, carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro,cyano, amino, carboxylic acid, carboxylic ester, carboxyl amide, reversecarboxyamide, substituted alkyl, substituted alkenyl, substitutedalkynyl, substituted carbocycle, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate; R2 is selected from H, F, Cl, Br, I,alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocycle, heteroaryl,formyl, nitro, cyano, amino, carboxylic acid, carboxylic ester, carboxylamide, reverse carboxyamide, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted carbocycle, substituted aryl,substituted heterocycle, substituted heteroaryl, phosphonic acid,phosphinic acid, phosphoramidate, phosphonic ester, phosphinic ester,ketone, substituted ketone, hydroxamic acid, N-substituted hydroxamicacid, O-substituted hydroxamate, N- and O-substituted hydroxamate,sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfonicacid, sulfonic ester, sulfonamide, N-substituted sulfonamide,N,N-disubstituted sulfonamide, boronic acid, boronic ester, azo,substituted azo, azido, nitroso, imino, substituted imino, oxime,substituted oxime, alkoxy, substituted alkoxy, aryloxy, substitutedaryloxy, thioether, substituted thioether, carbamate, substitutedcarbamate; R3 is selected from H, F, Cl, Br, I, alkyl, alkenyl, alkynyl,carbocycle, aryl, heterocycle, heteroaryl, formyl, nitro, cyano, amino,carboxylic acid, carboxylic ester, carboxyl amide, reverse carboxyamide,substituted alkyl, substituted alkenyl, substituted alkynyl, substitutedcarbocycle, substituted aryl, substituted heterocycle, substitutedheteroaryl, phosphonic acid, phosphinic acid, phosphoramidate,phosphonic ester, phosphinic ester, ketone, substituted ketone,hydroxamic acid, N-substituted hydroxamic acid, O-substitutedhydroxamate, N- and O-substituted hydroxamate, sulfoxide, substitutedsulfoxide, sulfone, substituted sulfone, sulfonic acid, sulfonic ester,sulfonamide, N-substituted sulfonamide, N,N-disubstituted sulfonamide,boronic acid, boronic ester, azo, substituted azo, azido, nitroso,imino, substituted imino, oxime, substituted oxime, alkoxy, substitutedalkoxy, aryloxy, substituted aryloxy, thioether, substituted thioether,carbamate, substituted carbamate; R4 is selected from H, F, Cl, Br, I,alkyl, alkenyl, alkynyl, carbocycle, aryl, heterocycle, heteroaryl,formyl, nitro, cyano, amino, carboxylic acid, carboxylic ester, carboxylamide, reverse carboxyamide, substituted alkyl, substituted alkenyl,substituted alkynyl, substituted carbocycle, substituted aryl,substituted heterocycle, substituted heteroaryl, phosphonic acid,phosphinic acid, phosphoramidate, phosphonic ester, phosphinic ester,ketone, substituted ketone, hydroxamic acid, N-substituted hydroxamicacid, O-substituted hydroxamate, N- and O-substituted hydroxamate,sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfonicacid, sulfonic ester, sulfonamide, N-substituted sulfonamide,N,N-disubstituted sulfonamide, boronic acid, boronic ester, azo,substituted azo, azido, nitroso, imino, substituted imino, oxime,substituted oxime, alkoxy, substituted alkoxy, aryloxy, substitutedaryloxy, thioether, substituted thioether, carbamate, substitutedcarbamate; and Cyc is an aryl, substituted aryl, heterocycle,substituted heterocycle, carbocycle, and substituted carbocycle.
 3. Acompound or a pharmaceutically acceptable salt thereof selected from thegroup consisting of:


4. A pharmaceutical formulation comprising a compound of claim 3 inassociation with a pharmaceutically acceptable carrier, diluent, orexcipient.